Patent Application: US-38694603-A

Abstract:
the composition as described serves for in vivo cartilage repair . it basically consists of a naturally derived osteoinductive and / or chondroinductive mixture of factors or of a synthetic mimic of such a mixture combined with a nanosphere delivery system . a preferred mixture of factors is the combination of factors isolated from bone , known as bp and described by poser and benedict . the nanosphere delivery system consists of nanospheres defined as polymer particles of less than 1000 nm in diameter in which nanospheres the combination of factors is encapsulated . the nano - spheres are loaded with the mixture of factors in a weight ratio of 0 . 001 to 17 % , preferably of 1 to 4 % and have a release profile with an initial burst of 10 to 20 % of the total load over the first 24 hours and a long time release of at least 0 . 1 per day during at least seven following days . the nanospheres are composed of e . g . - lactic acid / glycolic acid )- copolymer . the loaded nanospheres are e . g . made by phase inversion . the composition is advantageously utilized as a device comprising any biodegradable matrix in which the nanospheres loaded with the factor combination is contained .

Description:
[ 0033 ] fig1 shows a scanning electron micrograph of bp - loaded nanospheres . the microparticle sizes range from 100 - 1000 nm with the majority of individual particles ranging between 200 - 400 nm . the release rate profile of the inventive composition was determined by in vitro analysis of bp delivered from nanospheres . these nanospheres were made by phase inversion according to the method as disclosed by mathiowitz et al . ( nature 386 , 410 - 414 , 1997 ) of (( dl ) lactic acid / glycolic acid )- copolymer containing the two acids in a weight ratio of 50 : 50 and they were loaded with 1 % and with 4 % of bp . for determination of the release rate profile , the nanospheres were placed in a sterile saline solution and incubated at 37 ° c . bp released into the supernatant was measured using a bca assay ( pierce ). bp released from the nanospheres as specified shows two successive and distinct profile parts : a fast release ( initial burst ) of approximately 10 to 20 % of the loaded bp over the first 24 hours and a slow release of 0 . 1 to 1 % per day ( cumulative 40 % to 50 %) over 40 to 60 days ( fig2 ). the release is intermediate between zero - order and first - order kinetics . both the 1 % and 4 % encapsulated bp have similar release profiles . for attaining release rate profiles as specified above and as necessary for the improved results in cartilage repair the nanospheres are to be adapted accordingly when using factor mixtures other than bp . thereby , e . g the composition of the nanosphere copolymer , the molecular weight of the polymer molecules and / or the loading percentage of the nanospheres may be changed . the optimum nanosphere character for each specific case has to be found experimentally whereby the release rate profile is analyzed in vitro as described above . in the same way , the nanosphere delivery system can be modified regarding the percentage of bp to be released in the first 24 hours , percentage of bp to be released after 24 hours and / or length of time after the first 24 hours during which the remainder of bp is released . in addition , the percentage of bp loaded to the nanospheres is of course variable too , whereby for obtaining the results as described for the specified composition , all the modifications are to be chosen such that the resulting delivery keeps within the range as specified . all of the above parameters can be modified to account for the patient &# 39 ; s age , sex , diet , defect location , amount of blood present in the defect , and other clinical factors to provide optimal cartilage repair . for example , nanospheres with longer release rates are used for treating larger defects and / or for patients with fewer precursor cells ( e . g . older patients or patients with degenerative symptoms ). in contrast , patients with larger quantities of progenitor cells and / or smaller defects may require a shorter release rate profile . [ 0040 ] fig3 shows the release profile as shown in fig2 for nanospheres as specified above loaded with bp and with other proteins ( same loading percentages ) such as bsa ( bovine serum albumin ) or lysozyme . the drastically different release characteristics shows that the profile is dependent on the protein type also . the same is valid for a more hydrophobic mixture of bovine bone derived proteins ( pibp ). [ 0041 ] fig3 illustrates the singularity of the inventive combination consisting of the specific delivery system ( nanospheres as specified above encapsulating the factors ) and the specific protein mixture ( bp ) which is obviously the key to the improved results in cartilage repair as observed when using the inventive composition or device . to determine the length of time required for precursor cell repopulation of different sized defects , the following calculation was performed . we estimate that approximately 50 , 000 cells are recruited to the defect / day . since the cellular density of cartilage is about 4 × 10 7 cells / ml , a 10 μl volume defect will take approximately 8 days to fill with cells . fig4 plots the number of days required to fill different volume defects with cells . the figure assumes an infinite supply of cells and a constant rate of cell attraction to the defect site . the graph demonstrates that the larger a defect size is , the more time is required to completely fill it with cells . since a 60 μl volume defect will take over 45 days to fill , this figure demonstrates the necessity for a long term release of factors to induce differentiation of the precursor cells over up to a two month period . to determine whether bp bioactivity is harmed by the encapsulation process and to determine whether the released bp was fully bioactive , the following assay was performed . previously , it was demonstrated that 10t1 / 2 micromass cultures exposed to bp induce formation of a three dimensional spheroid structure that can be observed macroscopically in tissue culture wells ( atkinson et al ., j . cellular biochem . 65 : 325 - 339 , 1997 ). bp concentrations equal or greater than 20 ng / ml were required for spheroid formation . no spheroid forms in the absence of bp or at concentrations less than 10 ng / ml ( see following table ). in this assay , 10t1 / 2 mesenchymal stem cells act as in vitro models for the precursor cells recruited to a natural defect . we employed the same assay to test the bioactivity of bp released from 1 % loaded nanospheres . bp was eluted from nanospheres at 37 ° c . in a 5 % co 2 humidified incubator . after 24 hours 16 % bp is released ; and between 24 hours and 7 days , 7 % bp was released ( fig2 ). the supernatant was collected , serial dilutions were made , and the supernatant was added to 10t1 / 2 micromass cultures . bp released from nanospheres at both time points formed spheroids at concentrations greater than 20 ng / ml , but not at concentrations between 0 and 10 ng / ml ( see following table ). non - encapsulated bp also formed spheroids at concentrations greater than 20 ng / ml , but not at concentrations between 0 and 10 ng / ml . we conclude that both nanosphere encapsulation and , release of bp does not inhibit bp bioactivity . bp concentration ( ng / ml ) state of used bp 0 - 10 20 - 1000 non - encapsulated bp − + released from nanospheres ( 24 h ) − + released from nanospheres ( 168 h ) − + to determine the effect of bp slow release in the direct presence of micromass cultures , the following assay was performed . nanospheres were washed for 24 hours and the supernatant was discarded . the nanospheres were then added to micromass cultures at a quantity such that 10 or 25 ng / ml of bp would be released over 24 hours . release of 25 ng / ml resulted in spheroid formation whereas release of 10 ng / ml did not form spheroids ( fig5 ). similarly , the addition of 10 ng of non - encapsulated bp per ml did not form a spheroid whereas the addition of 25 ng of non - encapsulated bp per ml did form a spheroid . regarding the specific in vitro set - up , we conclude that slow release of bp over 24 hours is as effective as a single dose of bp . to determine whether the bp released from nanospheres was as chondrogenic as non - encapsulated bp , spheroids were analyzed for type ii collagen and proteoglycan content . 10t1 / 2 spheroids from the above assay that had formed with 1 μg of released bp per ml or 1 μg of non - encapsulated bp per ml were tested histologically with azure and h + e stains and immunocytochemically with antibodies to type ii collagen after 7 days . both encapsulated and non - encapsulated bp induced cartilage markers such as type ii collagen , proteoglycan , and round cell shape ( fig6 ). in addition , no qualitative differences were observed between encapsulated and non - encapsulated bp with respect to cell quantity , viability , morphology , or organization ( fig6 ). we conclude that bp retains full chondrogenic capacity after release from nanospheres . the in vitro models used for determining the chondroinductive effect of bp differ from the in vivo case by the fact that in the in vitro case the precursor cells are present in an appropriate number and in an appropriate distribution whereas in the in vivo case the precursor cells first have to populate the defect and for this reason have to migrate into the defect . only in the latter case and for achieving repair cartilage which resembles natural cartilage to a high degree , it is essential for the bp to be released over a prolonged time period according to a specific release profile . the following example shows that bp released from nanospheres induces cartilage repair in chondral defects in vivo whereby virtually all cells recruited to the defect become chondrocytes , whereby the cell structure obtained is ordered , and whereby a hyaline matrix is built up . using a sheep model , unilateral defects of 0 . 5 mm width , 0 . 5 mm depth and 8 to 10 mm length were created in the trochlear groove of the patella . the defects did not penetrate the subchondral bone . the sheep employed in this study were seven years old and displayed degenerative symptoms , including brittle bones , chondromalacia , and subchondral cysts . because of their advanced age and degenerative symptoms , these amimals probably have decreased numbers of precursor cells . the defects were then dressed according to hunziker and rosenberg ( j . bone joint surg . 78a ( 5 ): 721 - 733 , 1996 ) with minor changes . briefly , after enzymatic proteoglycan removal with chondroitinase ac , 2 . 5 μl of a solution containing 200 units thrombin per ml was placed in the defect . then , a paste was filled into the defect , the paste containing per ml : 60 mg sheep fibrinogen ( sigma ), 88 mg gelfoam ( upjohn ) and either 10 μg of bp - nanospheres or 10 μg of bp - nanospheres plus 80 ng rhigf - 1 ( r + d systems ). the nanospheres used were the nanospheres as specified in the description regarding fig2 and they were loaded with 1 % ( w / w ) of bp . assuming that the in vitro determined release rate is approximately the same as for the in vivo case , 10 to 20 ng bp per ml were released during the first 24 hours and approximately 0 . 1 to 1 ng per day for the following approximately 60 days . after eight weeks , necropsies were performed . the repaired cartilage histology showed that virtually all of the precursor cells were differentiated to chondrocytes throughout the defect . in addition , there was an ordered cartilage appearance with cells on the top being more flattened morphologically than cells in the center and with the presence of ordered , stacked chondrocytes in the lowest zone . the repaired cartilage was fully integrated into the endogenous tissue . in addition , the cartilage repaired with only bp - 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