Patent Application: US-8467702-A

Abstract:
a prosthesis or implant has a surface carrying a coating comprising at least one phospholipid at a concentration to improve osteointegration . a method of making such a prosthesis or implant and a kit comprising a prosthesis or implant is also described .

Description:
the examples below are in vitro experiments using simulated body fluids described in the literature and whose composition is given in examples 1 and 7 . various metal , ceramic and polymeric implant coupons have been coated with various phospholipid formulations . upon incubation of these coated coupons in the simulated body fluids , calcium phosphate deposition has been induced or enhanced . it is plausible that the same effect will occur in vivo and will result in improved osteointegration of an orthopaedic prosthesis or other implant . precipitation of calcium phosphate by phospholipid - containing vesicles in an aqueous environment thin films of excipients were produced on the surface of chromic acid cleaned round bottomed flasks from chloroform suspensions as well known to those skilled in the art . for the examples below , the following compositions were used : the thin films were resuspended in 2 ml of a simulated body fluid 11 ( 71 mm nacl ; 5 mm kcl ; 1 . 64 mm na 2 hpo 4 ; 2 . 36 mm cacl 2 and 50 mm tes buffer , ph 7 . 2 ). the final volume was adjusted , using the same buffer to give an eight - fold dilution of the original chloroform suspension . incubations were carried out in a shaking incubator set at 100 rpm and 37 ° c . in 12 ml polypropylene tubes . 1 ml samples were removed at daily intervals and washed three times in distilled water and fixed onto copper transmission electron microscopy ( tem ) grids using a 1 : 1 dilution with a 2 % ( v / v ) solution of ammonium molybdate . tem analysis of the samples demonstrated that for both the phosphatidylcholine : phosphatidylserine : cholesterol and phosphatidylcholine : phosphatidylinositol : cholesterol formulations , visible crystal - like structures were observed after 3 days incubation in the simulated body fluid . for the phosphatidylcholine : phosphatidylserine : phosphatidylinositol : cholesterol formulation , crystal structures were observed after 7 days incubation . fig1 shows a tem micrograph of a sample from the phosphatidylcholine : phosphatidylserine : cholesterol incubation and clearly demonstrates the presence of crystalline structures in intimate contact with the multi - lamellar vesicles ( large multi - layered liposomes ). two coating methodologies have been adopted for the surface coating of implant materials . these materials can be metal , ceramic or polymeric implant coupons . examples of the following have been used in the examples provided in this patent : three types of porous titanium oxide coupons ( disks of diameter 13 mm and 26 mm ; thickness 7 mm ) were provided by a commercial supplier of implant prostheses ; ti - pore300 ( porous surface with average pore diameter of 40 - 80 μm ), ti - pore600 . ( average pore diameter of 100 μm ). and ti - pore300 - ha ( the above ti - pore300 coupons plasma sprayed with a layer of the ceramic hydroxyapatite ( calcium phosphate )). titanium coupons were obtained from another supplier ( 13 mm diameter ; 6 mm thickness ) that had a flat surface morphology . polymeric materials were obtained form commercial suppliers and cast as known in the art to produce disks of implant material . the first coating methodology involves the dissolving of the excipients in hplc - grade chloroform . the formulations were applied either as 5 μl aliquots onto defined areas of the implant surface or as a greater volume to cover the entire implant surface . the chloroform quickly evaporates at room temperature , leaving the excipients in the form of a thin film . the second coating methodology involves the production of a phospholipid suspension in an aqueous media . the phospholipid was weighed out and dissolved in 100 μl hplc grade chloroform at a concentration of 222 μmol / ml . a thin film of phospholipid was produced on the wall of a glass round bottomed flask as described in example 1 . 1 ml of de - ionised water was then added stepwise to the flask and the flask shaken , resulting in a phospholipid suspension being produced . 40 μl of the phospholipid suspension was then gently layered onto a defined area of the material surface . the suspension was then dried onto the surface of the implant material by means such as air drying , freeze drying or rotary evaporation . this results in a thin film of phospholipid being deposited onto the surface of the material . the following spot sizes were produced on the ti - pore300 implant coupons . in the case of 444 μmol / ml solution , the spot concentration was estimated at 5 μmol / cm 2 . induction of calcium phosphate precipitation on the surface of a metal surgical implant using a phospholipid formulation containing phosphatidylserine this solution was then used to undertake a serial dilution to produce standards at 444 , 222 , 111 and 56 μmol / ml chloroform . 5 μl of each of the 4 standards were then applied onto distinct areas of a clean titanium implant coupon — ti - pore300 —( previously sonicated 3 times in hplc grade chloroform for 30 minutes ). the coupons were then placed in the incubation chamber and 10 ml of the simulated body fluid described in example 1 added . the samples were incubated at 37 ° c . for 7 days and daily visual checks undertaken . after two days incubation , white circles were visible on the titanium implant coupons at the points where the two highest excipient concentrations had been applied . the circle corresponding to the 444 μm / ml spot was approximately 1 - 2 mm in thickness at day 6 . at day 7 , photographs were taken of the titanium disc in situ prior to its removal from the incubation chamber . fig2 is of the surface of the ti - pore300 implant coupon whilst fig3 is a side on view of the 444 μm / ml coated spot . the implant coupons were rinsed 3 times in 10 ml of distilled water and placed in a drying oven at 60 ° c . for 18 hours . the surface of the implant coupon was then viewed by scanning electron microscopy and subjected to elemental dispersive x - ray analysis ( edax ), without coating the implant coupon with palladium . fig4 is an edax analysis undertaken on the uncoated ti surface and demonstrates no calcium or phosphorus present . this indicates that the ti surface itself does not act as a nucleating site for the precipitation of any significant amounts of calcium phosphate from the simulated body fluid used . fig5 is an edax analysis of the area coated with excipients at 444 μmol / ml . significant peaks for calcium and phosphorous were observed . the calcium peak can only be due to the presence of the deposited calcium whilst the phosphorus peak may be partially due to the presence of the phospholipids . edax spot analysis was also undertaken on the area coated with excipients at 222 μmol / ml and calcium and phosphorus peaks were again detected , demonstrating that a range of excipient concentrations are capable of inducing calcium precipitation onto the implant surface . the implant coupon was then washed three times using 20 ml hplc - grade chloroform for 30 minutes in a sonicating waterbath to remove the phospholipid . this cleaning protocol has previously been shown to remove phospholipid from the surface of the ti - pore300 implant coupon . the implant coupon was then subjected to further scanning electron microscopy analysis . fig6 is a scanning electron microscopy image of the surface previously coated with excipients at a concentration of 444 mmol / ml whilst fig7 is of the uncoated ti - pore300 surface . the difference in morphology is particularly striking . edax analysis was undertaken ( fig8 ) and demonstrates that calcium and phosphorus peaks were still present . it should be noted that there is also a peak for carbon present in the edax analysis ( fig8 ) which suggests that some of the phospholipid may still be bound to the surface in association with the calcium deposit . induction of calcium phosphate precipitation on the surface of a metal surgical implant using a phospholipid formulation containing phosphatidylinositol at day 7 , photographs were taken as in example 3 . fig9 is of the ti - pore300 implant coupon coated with the formulation containing phosphatidylinositol with the area corresponding to the 444 and 222 μm / ml spots to the forefront . the implant coupon was rinsed and dried as in example 3 . the surface of the implant coupon was then viewed by scanning electron microscopy and subjected to edax analysis . fig1 is an edax analysis of the area coated with excipients at 444 μmol / ml and demonstrates that the phosphatidylinositol formulation is also capable of binding calcium to the surface of the implant coupon . induction of calcium phosphate precipitation on the surface of a metal surgical implant using a phospholipid formulation of phosphatidylserine alone phosphatidylserine was weighed out and dissolved in hplc grade chloroform to give a final concentration of 222 μmol / ml . the experimental protocol used was as described in example 3 . the implant coupons were rinsed and dried as in example 3 . the surface of the implant coupon was then viewed by scanning electron microscopy and subjected to edax analysis . edax analysis of the area coated with excipients at 222 μmol / ml demonstrated that phosphatidylserine alone is also capable of binding calcium to the surface of the implant coupon . induction of calcium phosphate precipitation on the surface of a surgical implant using a formulation containing phosphatidylcholine after seven days incubation , a white circle became visible on the titanium implant coupon corresponding to where the excipients had been applied . the implant coupon was rinsed 3 times in 10 ml of distilled water and allowed to dry at room temperature for 4 days . fig1 is an edax analysis of the area coated with excipients and demonstrates that the phosphatidylcholine formulation is also capable of binding calcium to the surface of the implant coupon after 7 days . induction of calcium phosphate precipitation on the surface of a metal surgical implant using a different simulated body fluid the experimental procedure described in example 5 was repeated using a different simulated body fluid 12 ( 152 mm nacl ; 5 mm kcl ; 1 mm k 2 hpo 4 ; 1 . 5 mm mgcl 2 ; 27 mm nahco 3 ; 0 . 5 mm na 2 so 4 ; 2 . 6 mm cacl 2 and 50 mm tris buffer at ph 7 . 4 ). this simulated body fluid contains ions that are known to compete with calcium and phosphate in the precipitation of calcium phosphate in vivo . similar results were obtained after 7 days incubation in this simulated body fluid , demonstrating that the binding of calcium is not inhibited by the presence of competing divalent ions . induction of calcium phosphate precipitation on different metal surgical implant surfaces using a phospholipid formulation containing phosphatidylserine ti - pore600 implant coupons were coated with the phosphatidylserine formulation described in example 3 at 444 μmol / ml and incubated in 10 ml simulated body fluid at 37 ° c . photographs of the incubation chamber were taken at day 0 , 1 , 2 , 3 and 7 and scanning electron microscopy / edax analysis undertaken after 9 days incubation , again without coating the implant coupon with palladium . after only a few hours incubation , white circles corresponding to where the phospholipid formulation had been applied were visible . after 9 days incubation , the implant coupon was washed and dried as in example 5 . edax analysis did not detect any significant amounts of calcium or phosphorous on the uncoated surface as was the case for the ti - pore300 implant coupon . fig1 is an edax analysis of the area coated with excipients and demonstrates that the formulation is capable of binding calcium to the surface of different implant coupons . induction of calcium phosphate precipitation on different metal implant surfaces using a formulation containing phosphatidylserine only smooth titanium coupons were coated with phosphatidylserine only as described in example 5 at 222 μmol / ml . the experimental protocol used was as described in example 3 . after only a few minutes incubation , white circles corresponding to where the phospholipid formulation had been applied were visible . fig1 is an edax analysis of the area coated with excipients and demonstrates that the formulation is capable of binding calcium to the surface of different implant coupons . induction of calcium phosphate precipitation on ceramic surgical implant surfaces using a formulation containing phosphatidylserine ti - pore300 - ha implant coupons were coated with the phosphatidylserine formulation at 444 μmol / ml as described in example 3 and incubated in 10 ml simulated body fluid at 37 ° c . photographs were taken at day 0 , 1 , 2 , 3 and 7 . after only a few hours incubation , white circles corresponding to where the phospholipid formulation had been applied were visible . fig1 is the surface of the ti - pore300 - ha implant coupon after incubation for 2 hours in simulated body fluid demonstrating that a similar matrix can be formed on a ceramic surface . induction of calcium phosphate precipitation on a polymeric implant surface using a formulation containing phosphatidylserine only polymeric films of poly ( methyl methacrylate ) ( pmma ) were cast using techniques well known to those skilled in the art . the smooth polymeric films were then coated with a phosphatidylserine suspension in an aqueous medium as described in example 2 and incubations set up and analysed as described in example 8 . scanning electron microscopic analysis of the phospholipid coated poly ( methyl methacrylate ) films demonstrated a continuous thin coating on the surface ( fig1 ) prior to incubation in simulated body fluid . after incubation in simulated body fluid for 7 days , the scanning electron microscopic analysis demonstrated a that the coating of phospholipid had mineralised ( fig1 ) and scanning electron microscopy - edax analysis demonstrated the presence of calcium and phosphate crystals . surface mapping of the sample was also undertaken using scanning electron microscopy - edax analysis and it was found that the areas of the surface containing calcium and phosphate corresponded to the areas of the surface containing carbon , demonstrating that the calcium is associated with the areas of the polymer coated with phospholipid . induction of calcium phosphate precipitation on a metal surgical implant surface coated with a formulation containing phosphatidylserine , conditioned by incubation in serum a ti - pore300 implant coupon was coated with the phosphatidylserine formulation as described in example 3 . the implant coupon was then incubated for 1 hour in commercially available human serum at 37 ° c ., washed twice with 20 ml and then incubated in simulated body fluid as described in example 4 . scanning electron microscopy / edax analysis was then undertaken after 9 days incubation , again without coating the implant coupon with palladium . after approximately 30 minutes incubation in the simulated body fluid , a white circle corresponding to where the phospholipid formulation had been applied was visible . edax analysis of the area coated with excipients ( fig1 ) demonstrated that even in the presence of a conditioning layer of serum proteins , the formulation is capable of binding calcium to the surface of the implant coupon . the invention is not limited to the embodiments hereinbefore described which may be varied in both construction and detail . 1 . soballe k ., fiedman r . j . 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