Patent Application: US-201113640027-A

Abstract:
this invention provides a composition for controlled - release of polypeptide growth factors useful in tissue repair or engineering . the composition include a polypeptide growth factor covalently cross - linked to a biocompatible substrate by a transgultaminase . the cross - linking tethers the growth factor to a substrate so that it will not diffuse away from the desired site of application . it also concomitantly inactivates the growth factor . release and reactivation of the growth factor can be achieved by endogenously produced metalloproteinase or exogenously provided proteases such as collagenases . also provided are scaffolds , transplant devices , methods for using the same and methods for making the same ,

Description:
the growth factors are generally cross - linked to the carrier substrate by simply adding a suitable tgase to a mixture of the growth factors and the carrier substrate in a suitable medium under appropriate reaction conditions . for example , in an exemplary embodiment , microbial tgase was added to a blend of bsa and bmp2 to form bsa - bmp2 complexes . the reaction took place in a 1 % solution of bsa , bmp2 , pbs , and tgase in a ratio of 5 : 2 : 2 : 1 and was incubated overnight at room temperature . the bmp2 activity is de - activated in the resulting bmp2 - bsa complex . this complex form an exemplary delivery system of this invention and may be introduced to a tissue repair or regeneration site . once placed in the physiological environment , the complex will be broken down by enzymes in the native environment to release the bmp2 . the released bmp2 regains its activity and performs its bone induction function . tissue regeneration scaffolds may be similarly prepared to embed de - activated growth factors prior to use in repair sites . prepackaged kits of growth factors and tgases may also be beneficially prepared for commercial distribution . exemplary kits may include growth factor and tgases combinations suitable for a predetermined type of carrier substrate . these kits will have the advantage of providing convenience in both research and production settings . bmp - 2 is known to transdifferentiate a premyoblast c2c12 cell line by dose dependently increasing alkaline phosphatase ( alp ) activity . bmp - 2 alone ( fig1 a ), gelatin / bmp - 2 ( fig1 b ); and bmp - 2 treated with tgase ( fig1 f ) all induced alp activity at comparable levels . the high alp activity suggested that tgase or gelatin had no inhibitory effect on bmp - 2 activity . however , when bmp - 2 and gelatin mixture was treated with tgase , bmp - 2 activity was completely lost as exhibited by alp ( fig1 g ). this indicated that as bmp - 2 and gelatin bonded and formed a complex by the action of tgase , the formation of the complex ( gelatin - bmp - 2 complex ) shielded the bmp - 2 activity . significant differences were observed between gelatin - bmp - 2 complex and all other bmp - 2 containing samples ( p & lt ; 0 . 001 ) to determine whether bmp - 2 activity can be restored , gelatin - bmp - 2 complexes were digested by bacterial collagenase . as shown in fig2 , collagenase restored bmp - 2 activity ( fig2 d ) to a level that did not significantly differ from free bmp - 2 ( fig2 a ). collagenase itself showed no effect on bmp - 2 activity ( fig2 e ). this indicated that bmp - 2 was not only de - activated by tgase - gelatin crosslinking to gelatin , but that it could also be re - activated by collagenase digestion . to demonstrate controllable activation , various amounts of collagenase were added to each gelatin - bmp - 2 complex . fig3 exhibits an increased bmp - 2 activity as the dose of collagenase was increased ( r = 0 . 8870 , p & lt ; 0 . 001 ), showing that bmp - 2 can be re - activated by collagenase in a dose dependent manner . temporal effects of re - activation of bmp - 2 were evaluated by varying the incubation time using a defined concentration of collagenase . fig4 shows bmp - 2 activity to increase as incubation time was increased ( r = 0 . 7388 , p & lt ; 0 . 001 ), which demonstrates that a prolonged digestion results in increased bmp - 2 re - activation from the gelatin - bmp - 2 complex to elucidate the possible reaction mechanism , sds - page was used to monitor the protein complex formation and its digested byproducts after collagenase ( fig5 ). sds - page displayed bmp - 2 with a molecular weight of 26 kda in lane 2 , gelatin as band fragments of different molecular weight peptides between 37 kda and 116 kda in lane 3 and tgase as a single band with molecular weight around 37 kda in lane 4 . separate bands of bmp - 2 and gelatin were viewed in lane 6 indicating that no binding transpired between bmp - 2 and gelatin without tgase . the addition of tgase created a smear out of the fragmented gelatin peptide bands ( lane 7 ) suggesting that tgase generated the crosslinking among the gelatin fragments . the same smear was observed in lane 8 ( gelatin - bmp - 2 complex ), when bmp - 2 and gelatin underwent the reaction by tgase . collagenase alone was run in lane 5 but concentration was not high enough to be visualized on the gel . however , the concentration was enough to digest the gelatin and tgase crosslinked gelatin as evidenced by the absence of bands in lane 10 and lane 11 . the elimination of all bands of tgase crosslinked gelatin ( lane 10 ) and gelatin ( lane 11 ) show that collagenase completely digested gelatin and tgase crosslinked gelatin . however in lane 9 ( gelatin - bmp - 2 complex with collagenase ), collagenase digestion left behind a smear indicating components of the complex to be indigestible . in comparing bmp - 2 bands in lane 8 to lane 2 ( bmp - 2 alone ) or lane 6 ( gelatin / bmp - 2 ), a fading band density of bmp - 2 was observed . this suggested an uptake of bmp - 2 as it binds to gelatin . together with the effects associated with digestion , the smear on lane 9 most likely involved the bound bmp - 2 . furthermore , bmp - 2 in lane 12 showed no fragmentation or signs of digestion when treated with collagenase , suggesting bmp - 2 was the un - degradable component left behind in lane 9 . the smear also indicated that small undigested pieces of gelatin remained attached to bmp - 2 after collagenase treatment . to test the effects of mammalian collagenase activity on gelatin - bmp - 2 complex , mmps were obtained from rat skin . gelatin zymographs revealed that different types of mmps were expressed from the dermis rat skin layer after prolonged incubation periods in organ culture . signs of mmp2 production were observed from day 1 . mmp9 and mmp3 began to manifest from day 2 . all mmps were observed to increase with longer organ culture incubation ( fig6 a ); thus a gradient of mmps was created as daily collections from media were performed . for the re - activation of bmp - 2 , the gelatin - bmp - 2 complex was treated with a gradient of mmps . results show that native mammalian mmps re - activated bmp - 2 from the gelatin - bmp - 2 complex in a dose dependent manner ( fig6 b ). before conducting the in vivo bone induction test for the gelatin - bmp - 2 complex , samples of gelatin - bmp - 2 complex before lyophilization were taken and tested by the c2c12 cell based bmp - 2 activity assay . the results confirmed that bmp - 2 was completely de - activated and that collagenase digestion of the samples led to re - activation ( data not shown ). gelatin crosslinked by tgase was co - lyophilized with a pre - fowled gelatin - bmp - 2 complex before implantation . after 35 days of implantation in the abdominal muscle pouch , samples were explanted and stained . h & amp ; e staining showed active cellular infiltration around and inside all implants . for scaffold alone , we observed it to be partially degraded with fibrous tissue ingrowths and no bone formation ( fig7 a ). however , when implants were supplemented with gelatin - bmp - 2 complex , new bone formation was clearly evident . interestingly , areas of new bone formation were focused at the outer edge of the implant ( fig7 b ). the lack of bone formation in the center part of implant indicated that cell - instructive activation only occurred on the surface of the construct under the current design and time course of the study . the results of alp activity from explants correlated with the histological findings . gelatin - bmp - 2 complex group exhibited higher levels of alp activity compared to scaffold alone ( fig7 c ), indicating active bone formation in the gelatin - bmp - 2 complex containing explants . to demonstrate controllable re - activation with another enzyme other than collagenase , various amounts of trypsin were added to each gelatin - bmp - 2 complex . fig9 exhibits an increased bmp - 2 activity as the dose of trypsin was increased , showing that bmp - 2 can be re - activated by collagenase in a dose dependent manner . temporal effects of re - activation of bmp - 2 were also evaluated by varying the incubation time using a defined concentration of trypsin . fig9 shows bmp - 2 activity to increase as incubation time was increased , which demonstrates that a prolonged digestion results in increased bmp - 2 re - activation from the gelatin - bmp - 2 complex . bmp - 2 is de - activated when bound to bsa and re - activated with trypsin to confirm bsa &# 39 ; s role in the de - activation of bmp - 2 , bsa - bmp - 2 complexes were prepared and tested on the c2c12 alp assay . samples of bmp - 2 alone and bsa mixed with bmp - 2 were also taken as positive controls . for re - activation , one sample of the bsa - bmp - 2 complex was incubated with 1 u / ml of collagenase and another with 0 . 025 % concentration of trypsin for 1 hour at 37 ° c . in fig1 , the formation of bsa - bmp - 2 complex resulted in the de - activation the bmp - 2 activity . however , unlike the gelatin based complex , collagenase did not restore the bmp - 2 &# 39 ; s activity . bmp - 2 activity was re - activated after the adding the trypsin to the bsa - bmp - 2 complex . because bsa is digested by trypsin and not by collagenase , this results further indicates that the protein that binds to the bmp - 2 can selectively re - activate bmp - 2 depending on the proteases it reacts with . bmp - 2 is de - activated when bound to bsa and re - activated with trypsin in the previous study , bmp - 2 activity was re - activated by the addition of collagenase . in this study , other proteases were sought out and incubated with the gelatin - bmp2 and the bsa - bmp - 2 complexes , bmp - 2 activity was determined to observe if bmp - 2 could be re - activated by proteases such as pronase , collagenase , trypsin , chymotrypsin , papain , and chymopapain . for gelatin - bmp2 - tgase , higher concentration of trypsin and collagenase was shown to re - activate more bmp - 2 activity for gelatin - bmp - 2 - tgase complex in fig1 , pronase and chymotrypsin also showed a controllable re - activation of bmp - 2 activity . although the chymopapain and papain show low bmp - 2 activity , later studies have indicated that the protease digests bmp - 2 . lower dosages of chymopapain and papain may show protective properties of the complex . for bsa - bmp - 2 complex , results indicated that bmp - 2 could also be controlled and reactivated by other proteases ( fig1 ). similarily with gelatin - bmp - 2 complex , chymopapain and papain did not re - activate bmp - 2 from the bsa - bmp2 complex . chymotrypsin , trypsin and pronase showed re - activity of bmp - 2 and dosage control . again , the result repeatedly showed that unlike gelatin - bmp2 complex , collagenase did not re - activate bmp - 2 from bsa - bmp - 2 complex . in observing pronase digestion on both gelatin and bsa complexes , bmp - 2 activity was re - activated as the concentration of pronase were increased , ( fig1 ) however , when unbound bmp - 2 was added to pronase , the bmp - 2 activity decreased as the added pronase concentration increased ( fig1 ). this suggests bmp - 2 is digested by pronase . pronase is relatively non - selective in digestion of proteins and can digest many types of proteins including bmp - 2 , bsa and gelatin taken together , the results suggest that the bsa or gelatin protein may have protected the bmp - 2 by having the carrier being digested first . the carrier substrate not only have a role as switch but also act as a protectant of bmp - 2 . in a previous study ( kuwahara , yang et al ., 2010 ), mscs were successfully encapsulated by gelatin - tgase and delivered to repair sites . msc in this study was replaced with c2c12 to better assess bmp - 2 activity by alp activity . gelatin - bmp2 complex was made separately with 2 % gelatin solution , producing a liquid - like consistency . aliquots of gelatin - bmp - 2 complex with bmp - 2 at 4 ng / ul and 10 ng / ul concentration were tested and was shown to be de - activated . the gelatin - bmp2 complex was also subjected to collagenase and was shown to re - activate bmp - 2 . when c2c12 were encapsulated in a 10 % gelatin crosslinked with tgase , the gelatin - bmp - 2 complex was also included in the mix . the gelatin - tgase gel containing the gelatin - bmp - 2 complex and c2c12 were incubated in wells . the surrounding media of the gelatin - tgase gels was taken daily for detecting digestive enzymes released by c2c12 . the zymograph results show that mmp2 was released daily from c2c12 ( fig1 ), this suggests the mmp2 released by the c2c12 can re - activate the bmp - 2 in the gelatin - bmp - 2 complex . alp activity from the harvested c2c12 showed that c2c12 was able to activate alp activity from the bmp - 2 that was bound in the gelatin - bmp - 2 complex ( fig1 ). alp activity started to increase after the third day for the c2c12 encapsulated containing 10 ng / ul bmp2 and the fourth day for the 4 ng / ul bmp2 . the c2c12 with no gelatin - bmp2 - tgase had no alp activity . the alp activity of the encapsulated c2c12 increased , as the encapsulation time for c2c12 became longer . taken together with the mmp2 released by c2c12 ( fig1 ), c2c12 differentiated toward a osteoblastic lineage as the mmps released by c2c12 were re - activating the bmp - 2 . immobilization of soluble regulatory peptides on carrier molecules or scaffolds which are later released by cellular activation may provide a powerful means to control cell behavior and enable complex processes of tissue formation and regeneration . the examples described herein is the first report of the novel discovery that the activity of growth factor when conjugated to a carrier protein by enzymatic action can be switched off converting from an active ligand into its latent form . surprisingly , through the action of proteolytic action such as mmps , the growth factor can be re - activated into its full potential . schematic mechanism is illustrated in fig8 . this new drug delivery method by immobilization of bioactive ligand onto a carrier molecule not only changes its biomolecular characteristics ( active - latent - active ) but also creates signal controlling microenvironment by cell matrix interaction . embodiments of the present invention uses a native form of growth factor . in contrast , all previously known method of enzymatically regulated release of growth factors requires the engineering of new fusion proteins that contain exogenous substrates for enzymatic crosslinking and / or for release by enzymatic degradation . in addition to the extra protein expression work , utilizing structurally modified recombinant proteins may be of concern for clinical applications . embodiments of the present invention clearly demonstrates that the shielding of growth factor activity from cells can be achieved through binding a soluble biomaterial to the growth factor at the molecular level . while not intending to be bound by any particular theory , we hypothesize that the active sites of the growth factors are masked with protective peptide , gelatin , upon cross - linking . this contrasts with the traditional methods of using solid or semi - solid biomaterials for creating a separation barrier between the growth factor and cells . the tgase exhibits its effect by covalently binding the ε - amino group of a lysine residue to the γ - carboxamide group of a glutamine . however , for most tgase protein reactions , tgase was found to be highly selective towards only one single or rather few glutamines ( q ) among the many other glutamine residues present in proteins . the substrate reactivity toward tgase is determined by the amino acids surrounding the glutamine in the peptide chain . repeated glutamines were shown to increase reactivity with tgase . ohtsuka et al . reported that the substrate reactivity is enhanced when the leucine ( l ), glutamic acid ( e ) or valine ( v ) is placed in front of the q . ito et al . enhanced microbial tgase crosslinking of rgd to gelatin by synthesizing a peptide with the ls in front the q as seen in the resulting sequence rgdllq . tgase also shows reactivity preference toward different lysines depending on their location or sequence . if growth factors have endogenous reactive sites for tgase to bind to , the growth factor can be tethered onto a suitable reactive scaffold like gelatin or collagen which contain both reactive lysine and glutamine residues . in embodiments of this invention , unmodified native bmp - 2 was crosslinked directly onto gelatin , revealing that endogenous tgase reaction sites are available on bmp - 2 . the de - activation of the growth factor as a result of this crosslinking reaction was initially discouraging , but the later re - activation of bmp - 2 after the addition of collagenase brought to light an interesting phenomenon . because collagenase was found to be selective towards digesting both tgase crosslinked and non - crosslinked gelatin and had no effect on bmp - 2 , gelatin appears to be responsible for de - activation . although the binding sites where gelatin attaches onto bmp - 2 still need to be elucidated , they do not seem to be binding to the active sites of bmp - 2 . covalent bonds between ε - amino group and γ - carboxamide group are not digestible by collagenase , and if the bonds occurred at the bmp - 2 active site , the residual gelatin fragment after digestion would have blocked the bmp - 2 active site permanently . biological active regions of bmp - 2 are reported to be located on the two epitopes , the wrist epitope and knuckle epitope . the wrist epitope associates and binds with the bmp receptor ia and the knuckle epitope associates with the bmp receptor type ii . it has been shown that mutating regions of one or the other epitope of bmp - 2 reduces but not entirely demolishes the biological effect of bmp - 2 . in embodiments of this invention , bmp - 2 activity in the gelatin - bmp - 2 complex was completely lost . since c2c12 cells have both bmp receptor type ia and ii , it is possible that the bound gelatin , which serves as a latent sequence of bmp - 2 , shielded both bmp - 2 epitopes that binds to the c2c12 cell receptors . although bmp - 2 activity assays and protein chromatography seems to support this pathway , the detailed mechanism requires further investigation . construction of mmp - sensitive biomaterial for guided tissue repair has been drawn a lot of attention in recent years . as a denatured form of collagen , gelatin is susceptible to degradation by various proteolytic enzymes , including mmps . in our study , we demonstrated that , besides bacterial collagenase , gelatin - bmp - 2 complex can be readily activated by tissue derived mmps . mmps are usually released by cells at the defect site for purposes of tissue repair and remodeling . besides their role in remodeling , mmps affect other cell functions such as proliferation and apoptosis . secreted as inactive proenzymes and activated near the cell surface or expressed at the surface in activated form as membrane - type mmps ( mt - mmp ), these enzymes can cleave virtually all constituents of the ecm . in exemplary embodiments this invention , ecm derived collagen served dual roles , both as scaffold and growth factor switch . our implant data clearly demonstrates that a tgase crosslinked gelatin scaffold can induce active cell infiltration and regeneration as well as support new bone formation . overall , the multistep osteoinductive events can be described as follows : bmp - 2 is covalently immobilized and the bioactive osteoinductive signal switched off in the gelatin matrix when implanted . the mmp - sensitive tgase crosslinked gelatin scaffold guides the inflammatory and osteoprogenitor cells to migrate into the matrix . the cells produced the mmps which in addition to degrading the matrix released bmp - 2 into its active form . bmp - 2 &# 39 ; s osteoinductive signaling causes the differentiation of the osteoprogenitor cells in this microenvironment into osteoblasts . gelatin ( type b 225 bloom , sigma aldrich ) was dissolved and autoclaved in distilled water to make a 10 % gelatin stock . the autoclaved gelatin was aliquoted and stored at 4 ° c . until use . a 2 % percent gelatin solution was made by diluting from a 10 % gelatin stock at 37 ° c . with bmp - 2 buffer ( 25 mm tricine , ph 7 . 2 , 15 mm sucrose , 1 . 7 mm nacl , and 0 . 01 % tween 80 ). microbial transglutaminase ( activa ti ajinomoto , japan , tgase ) from streptomyces mobaraense was purified using a sepharose fast flow column [ 47 ]. briefly , 3 g of crude tgase were dissolved in a phosphate buffer ( 20 mm phosphate and 2 mm edta , ph 6 . 0 ) and gently mixed with 3 ml of pre - equilibrated s sepharose fast flow beads ( sigma ). after incubation at 4 ° c . overnight with occasional vortexing , the protein solution and beads mixture were batch loaded into a column . after washing with 4 volumes of phosphate buffer , tgase was eluted with eluting buffer ( phosphate buffer with 800 mm nacl ). protein concentration was monitored by the bradford method ( bio - rad ) utilizing bsa as a standard . bmp - 2 ( r & amp ; d systems ) was kept in stock concentrations of 20 ng / μl at − 20 ° c . in buffer solution ( 5 mm glutamic acid , 2 . 5 % glycine , 0 . 5 % sucrose , and 0 . 01 % tween 80 ). gelatin / bmp - 2 : two percent gelatin was mixed with 20 ng / μl bmp - 2 stock at a ratio of 5 : 2 at room temperature . the final bmp - 2 concentration was 4 ng / μl . gelatin - bmp - 2 complex : the covalent binding of bmp - 2 to gelatin was prepared using tgase . tgase was added to the gelatin / bmp - 2 mixture at a final concentration of 25 μg / ml . the reaction was carried out at room temperature for 18 hours . mixtures were either prepared fresh or stored at − 80 ° c . before determining bmp - 2 activity by using the c2c12 cell assay . gelatin - bmp - 2 complex was treated with collagenase to evaluate bmp - 2 release and its activation profile . the complex was treated with 1 u / ml ( final ) bacterial collagenase ( 190 u / mg , type 2 , worthington ) at 37 ° c . for 1 hour . samples were collected and taken for the c2c12 cell based bmp - 2 activity assay . for the dose response study , the gelatin - bmp - 2 complex was treated with collagenase ( 0 . 2 - 2 u / ml for 1 hour at 37 ° c .) before samples were taken to the c2c12 cell based bmp - 2 activity assay . for the time course study , a final concentration of 1 u / ml collagenase was added to separate aliquots of the gelatin - bmp - 2 complex solution . the solution was incubated at 37 ° c . and retrieved at various time points from 0 to 180 minutes , and stored at − 80 ° c . before assaying for bmp - 2 activity . to examine the bmp - 2 interaction with gelatin , sample concentrations were increased because concentrations used in the c2c12 cell based bmp - 2 activity assay were too low to be visible on sds - page . the final concentrations of bmp - 2 and tgase in the mixtures were raised to 100 ng / μl and 40 μg / ml respectively . gelatin ( 1 mg / ml ) was generated from type i from rat tail collagen ( lab prepared ) by heat denaturation at 55 ° c . for 4 hours . collagenase final concentration was 1 . 5 u / ml . reactants were mixed at 1 : 1 ( v / v ) with sds sample buffer ( 125 mm tris ph 6 . 8 , 2 % sds , 0 . 1 % bromophenol blue , and 25 % glycerol ) and heated at 100 ° c . for 5 min prior to loading onto the 3 - 18 % gradient sds - page gel . the samples underwent electrophoresis at 90 v until the frontier reached the end of the gel . the gels were stained with coomassie blue solution ( 62 . 5 % methanol , 25 % acetic acid and 0 . 125 % coomassie blue r250 ( bio - rad )) overnight and destained with 30 % methanol and 1 % formic acid for 5 hours . within two hour after the euthanization of a fisher 344 rat , a 2 cm × 2 cm square piece of skin was removed from the abdominal area after being shaved and the surface - treated with 70 % ethanol . the dermis was separated from the keratin layer and the attached muscle with a scalpel . the dermis was incubated in pbs containing a 2 % antibiotic - antimycotic solution ( mediatech ) at 4 ° c . for 15 hours . the skin was subsequently rinsed twice with pbs , excised into 0 . 5 cm × 0 . 5 cm pieces and placed into a 60 mm tissue culture plate with 5 ml of dmem containing 10 ng / ml ( final ) of tnfα ( r & amp ; d systems ). aliquots of 200 μl were collected after 1 , 2 , 3 , 4 and 5 days of incubation at 37 ° c . 5 % co 2 . mmps were measured by a gelatinolytic zymograph . samples of 20 μl from the collected organ culture media were added to each of 100 μl aliquots of gelatin - bmp - 2 complex and incubated at 37 ° c . for 1 hour . a subsequent c2c12 cell based bmp - 2 activity assay was conducted on the gelatin - bmp - 2 complex solution treated with organ culture media . collected organ culture media without gelatin - bmp - 2 complex was also assayed for bmp - 2 activity as a control . media collected from the organ culture were analyzed for mmp activity through a gelatin zymograph as described [ 48 ]. in short , the culture media was mixed with sample buffer at a 1 : 1 ratio ( 250 mm tris ph 6 . 8 , 10 % sds , 50 % glycerol , 2 . 5 mg / ml of bromophenol blue ) without reducing agent or heating . the sample was loaded into a gelatin ( 5 . 50 □ mg / ml ) containing 10 % acrylamide / biascrylamide ( bio - rad ) and underwent electrophoresis with constant voltage ( 90 v ) for 5 . 5 hours . afterwards , the gel was washed with 2 . 5 % triton x - 100 to remove the sds , rinsed with 500 mm tris - hcl ph 7 . 5 , and then incubated overnight at 37 ° c . with the developing buffer ( 50 mm tris ph 7 . 5 , 5 mm cacl 2 , and 200 mm nacl ) for 16 hours . the zymographic activities were revealed by 1 hour staining with coomassie blue staining solution and subsequent overnight destaining with 30 % methanol and 1 % formic acid . bmp - 2 dose dependently induces alkaline phosphatase ( alp ) activity in a c2c12 mouse myoblast cell line [ 5 ]; therefore , activities of sequestered and released bmp - 2 can be determined by alp assay with c2c12 cells . c2c12 cells ( atcc ) were seeded onto a 96 - well - plate at a concentration of 1 . 25 × 10 4 cells per well with 100 μl of 10 % fbs / dmem . the plate was incubated at 37 ° c ., 5 % co 2 overnight for attachment . the media was exchanged to test media with 200 μl of 1 % fbs / dmem . aliquots of 10 μl samples were added to each well and incubated for 48 hours . at the end of the incubation period , the media was removed , and the cells were washed twice with cold pbs . the cells were then lysed by adding 30 μl of 0 . 5 % triton x - 100 / pbs and undergoing three freeze / thaw cycles . for pnpp substrate solution , each pnpp tablet ( 5 mg p - nitrophenyl phosphate disodium salt / tablet , thermo scientific ) was dissolved in 5 ml diethanolamine buffer ( 1 . 02 m diethanolamine 0 . 5 mm mgcl 2 ph 9 . 8 ). one hundred pi of pnpp substrate solution were added to the cell lysates and incubated at 37 ° c . for 30 min . alp activity was determined by recording absorbance at 405 nm and normalized by protein content using a rca protein assay kit ( bio - rad ). each gelatin - bmp - 2 complex sample destined to be implanted was prepared by mixing 3 μg of bmp - 2 ( 30 μl ) into a 200 μl of 5 % gelatin solution and incubated with tgase ( 25 μg / ml , final concentration ) overnight . before lyophilization , aliquots of the mixtures , with or without collagenase digestion , were assayed in vitro with c2c12 cells for bmp - 2 activity to confirm bmp - 2 binding and release . lyophilized 5 % gelatin gels that were crosslinked with 25 μg / ml of tgase were used as controls . animal use protocols were approved by iacuc of the university of southern california . a total of 6 fisher 344 rats ( male , 8 weeks , wt 190 - 210 g ) were used in the in vivo study . muscle pouches were created in the abdominal muscles bilaterally at 6 sites by sharp and blunt dissection and subsequently packed with lyophilized gels . samples were harvested after 35 days . each sample was divided into two , one half fixed in 10 % neutral buffered formalin for histology ( h & amp ; e ) and the other half homogenized in 0 . 5 % triton x - 100 for alp activity using p - nitrophenyl - phosphate ( pnpp ) as a substrate . absorbance was measured at 405 nm after 15 min of incubation at 37 ° c . the activity was normalized by total protein content using a bca protein assay kit ( bio - rad ). the student t - test was performed to evaluate differences in all c2c12 cell based bmp - 2 activity assays . data were expressed as mean ± sd . the pearson correlation test was used for evaluating the time and dose dependent effects of collagenase digestion . statistical significance was set at p & lt ; 0 . 05 in all analyses . although the present invention has been described in terms of specific exemplary embodiments and examples , it will be appreciated that the embodiments disclosed herein are for illustrative purposes only and various modifications and alterations might be made by those skilled in the art without departing from the spirit and scope of the invention as set forth in the following claims . 1 . kugimiya , f ., et al ., involvement of endogenous bone morphogenetic protein ( bmp ) 2 and bmp6 in bone formation . j biol chem , 2005 . 280 ( 42 ): p . 35704 - 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