Abstract:
An osteoinductive bone graft substitute including an osteoinductive calcium phosphate component retained within a collagen scaffold, wherein the collagen scaffold is formulated to maintain the osteoinductivity of the calcium phosphate component while also exhibiting the following characteristics (1) ability to absorb bone marrow and/or other physiologic liquids, (2) compression resistance; (3) moldability; (4) retaining the calcium phosphate component during and after manipulation of the collagen scaffold and (5) fibrous collagen matrix that retains but does not occlude the osteoinductive calcium phosphate component.

Description:
CROSS REFERENCE TO RELATED APPLICATIONS 
     This application is a continuation of U.S. patent application Ser. No. 14/059,437, filed on Oct. 21, 2013, which claims the benefit of the filing date of U.S. Provisional Patent Application No. 61/716,428 which was filed on Oct. 19, 2012. The content of U.S. application Nos. 14/059,437 and 61/716,428 is incorporated by reference as part of this application. 
    
    
     FIELD 
     This application relates generally to bone graft substitutes and more specifically toward an osteoinductive bone graft substitute comprising a calcium phosphate component and a collagen matrix. 
     SUMMARY 
     The present application is directed toward an osteoinductive bone graft substitute comprising a calcium phosphate component and a collagen matrix. The osteoinductive bone graft substitute includes an osteoinductive calcium phosphate component retained within a collagen scaffold, wherein the collagen scaffold is formulated to maintain the osteoinductivity of the calcium phosphate component while also exhibiting the following characteristics (1) ability to absorb bone marrow and/or other physiologic liquids, (2) compression resistance; (3) moldability; (4) retaining the calcium phosphate component during and after manipulation of the collagen scaffold and (5) fibrous collagen matrix that retains but does not occlude the osteoinductive calcium phosphate component. The osteoinductive bone graft substitute described herein is greater than 70% porous and is inherently osteoinductive (i.e. it does not require the addition of bone marrow aspirate to be osteoinductive). 
    
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
         FIG. 1  is a scanning electron micrograph of calcium phosphate granules according to a first embodiment of the bone graft substitute; 
         FIG. 2  is a scanning electron micrograph of a bone graft substitute comprising the calcium phosphate granules of  FIG. 1  and a collagen matrix; 
         FIG. 3  is a scanning electron micrograph of calcium phosphate granules according to an alternative embodiment of the bone graft substitute; 
         FIG. 4  is a scanning electron micrograph of a bone graft substitute comprising the calcium phosphate granules of  FIG. 3  and a collagen matrix; 
         FIG. 5  is a scanning electron micrograph of calcium phosphate granules according to another alternative embodiment of the bone graft substitute; and 
         FIG. 6  is a scanning electron micrograph of a bone graft substitute comprising the calcium phosphate granules of  FIG. 5  and a collagen matrix. 
     
    
    
     DETAILED DESCRIPTION 
     According to an exemplary embodiment, the calcium phosphate component of the osteoinductive bone graft substitute is an osteoinductive calcium phosphate, such as the calcium phosphate described in U.S. Pat. No. 7,942,934, which is hereby incorporated by reference in its entirety. However, other osteoinductive calcium phosphates may be also be used with the collagen matrix formulation described in the examples herein to achieve an osteoinductive bone graft substitute. 
     EXAMPLE 1 
     Handling Characteristics of Scaffold Prototypes 
     Preparation of the Materials 
     Five bone graft substitute prototypes were manufactured according to different formulations using variable collagen compositions, collagen and granules concentrations and crosslinking times, as shown in Table 1. The granule particle size range was between 500 μm and 1000 μm. Lyophilization was followed by dihydrothermal (DHT) crosslinking treatment in all samples. Lyophilization was performed, according to Table 2. DHT temperature was 110° C. for all formulations. All prototypes were gamma sterilized (25-30 kGy). After sterilization, each prototype was hydrated in preparation of handling evaluation. Hydration of prototypes was performed by placing each prototype in a petri dish and soaking with 5 cc of heparinized sheep blood. 
     
       
         
               
             
               
               
               
               
               
               
               
               
             
           
               
                 TABLE 1 
               
             
             
               
                   
               
               
                 Formulation Table 
               
             
          
           
               
                   
                   
                   
                 Collagen 
                 Granule 
                 Collagen 
                   
                   
               
               
                   
                 Collagen 
                 Conc 
                 Final  
                 Final 
                 Slurry 
                 Granule 
                 DHT 
               
               
                 Prototype ID 
                 Composition 
                 (mg/ml) 
                 wt % 
                 wt % 
                 (ml) 
                 (gr)  
                 (days) 
               
               
                   
               
               
                 Formulation 1 
                 Acid 50%/ 
                 65 
                 16 
                 84 
                 75 
                 25 
                 1 
               
               
                   
                 Pepsin 50% 
                   
                   
                   
                   
                   
                   
               
               
                 Formulation 2 
                 Acid 80%/ 
                 65 
                 16 
                 84 
                 75 
                 25 
                 1 
               
               
                   
                 Pepsin 20% 
                   
                   
                   
                   
                   
                   
               
               
                 Formulation 3 
                 Acid 
                 30 
                 20 
                 80 
                 89 
                 11 
                 0 
               
               
                   
                 Swollen 
                   
                   
                   
                   
                   
                   
               
               
                 Formulation 4 
                 Acid 
                 50 
                 12 
                 88 
                 73 
                 27 
                 0 
               
               
                   
                 Swollen 
                   
                   
                   
                   
                   
                   
               
               
                 Formulation 5 
                 Pepsin 
                 65 
                 16 
                 84 
                 75 
                 25 
                 3 
               
               
                   
                 Treated 
               
               
                   
               
             
          
         
       
     
                                                         TABLE 2                   Lyophilization Program                Temp (° C.)   Vacuum (mT)   Time (min)                    Initial   −50   100   30       Phase I   −35   200   120           −25   200   360           −18   200   480           −10   200   480           −6   200   480           0   200   720       Phase II   25   200   600                    
Physical Handling of the Samples
 
     In order to determine the optimal prototype formulation, each prototype was evaluated based on its response to physical handling. Prototype samples were bended, twisted, pulled and gently compressed to simulate handling conditions. Handling properties which were evaluated included compression resistance when wet, cohesiveness, moldability, flexibility and strength. 
     Handling properties of the prototypes were evaluated on a score from one (1) to five (5), with 1 being “poor” and 5 being “excellent.” Each prototype was evaluated three (3) times, each time by a different expert user. 
     Results of Physical Handling 
     Results of the physical handling of each prototype are shown in Tables 3 and 4. 
     
       
         
               
             
               
               
               
               
               
               
             
           
               
                 TABLE 3 
               
             
             
               
                   
               
               
                 Handling Properties - Average Rating 
               
             
          
           
               
                   
                 Compression 
                   
                   
                   
                   
               
               
                 Prototype 
                 Resistance 
                   
                   
                 Flexi- 
                   
               
               
                 ID 
                 (Wet) 
                 Cohesiveness 
                 Moldability 
                 bility 
                 Strength 
               
               
                   
               
               
                 Formu- 
                 4 
                 4 
                 3 
                 3 
                 4 
               
               
                 lation 1 
                   
                   
                   
                   
                   
               
               
                 Formu- 
                 4 
                 4 
                 3 
                 3 
                 4 
               
               
                 lation 2 
                   
                   
                   
                   
                   
               
               
                 Formu- 
                 2 
                 5 
                 5 
                 2 
                 1 
               
               
                 lation 3 
                   
                   
                   
                   
                   
               
               
                 Formu- 
                 2 
                 3 
                 2 
                 2 
                 2 
               
               
                 lation 4 
                   
                   
                   
                   
                   
               
               
                 Formu- 
                 5 
                 4 
                 3 
                 4 
                 5 
               
               
                 lation 5 
               
               
                   
               
               
                 1 = poor; 2 = below average; 3 = average; 4 = above average; 5 = excellent 
               
             
          
         
       
     
     
       
         
               
             
               
               
             
           
               
                 TABLE 4 
               
             
             
               
                   
               
               
                 Results of Physical Handling 
               
             
          
           
               
                 Prototype 
                   
               
               
                 ID 
                 Comments 
               
               
                   
               
               
                 Formulation 
                 Dry: There were some flakes in the vial 
               
               
                 1 
                 Wet: Soaked up more than 95% of 5 cc blood.  
               
               
                   
                 Minimum granule loss when manipulated. Compression 
               
               
                   
                 resistant. After manipulating, bounces back to its 
               
               
                   
                 original shape (good shape memory property). 
               
               
                   
                 Bends well. 
               
               
                 Formulation 
                 Dry: There were some flakes in the vial 
               
               
                 2 
                 Wet: Soaked up more than 95% of 5 cc blood.  
               
               
                   
                 Minimum granule loss when manipulated. Compression 
               
               
                   
                 resistant. After manipulating, bounces back to its 
               
               
                   
                 original shape (good shape memory property). 
               
               
                   
                 Bends well. Similar handling characteristics 
               
               
                   
                 as Formulation 1 
               
               
                 Formulation 
                 Dry: There were minimum flakes in the vial. Felt lighter, 
               
               
                 3 
                 softer and fluffier compared to Formulations 1, 2, 4 
               
               
                   
                 and 5. No visible granules in the scaffold. 
               
               
                   
                 Wet: Soaked 50% of 5 cc blood and shrunk. Turned in  
               
               
                   
                 to putty. No particle loss during handling. Can be 
               
               
                   
                 shaped in to a ball, rod, taco and other different 
               
               
                   
                 shapes. Excellent putty. 
               
               
                 Formulation 
                 Dry: There were some flakes in the vial 
               
               
                 4 
                 Wet: Soaked up more than 90% of 5 cc blood. When  
               
               
                   
                 manipulated falls apart and losses granules. Did not meet 
               
               
                   
                 minimum expectations for handling characteristics. 
               
               
                 Formulation 
                 Dry: There were some flakes in the vial 
               
               
                 5 
                 Wet: Soaked up more than 95% of 5 cc blood. Minimum  
               
               
                   
                 granule loss when manipulated. Compression resistant. 
               
               
                   
                 After manipulating, bounces back to its original shape 
               
               
                   
                 (good shape memory property). Bends well. Performed 
               
               
                   
                 better than the other formulations. 
               
               
                   
               
             
          
         
       
     
     According to the results, Formulation 5 performed the best as a compression resistant matrix. This was followed by Formulation 1 and Formulation 2 (both showed similar handling properties). Formulation 3 acted as putty and is an excellent candidate for use in putty form. Formulation 4 did not exhibit desired handling properties. 
     EXAMPLE 2 
     Optimization of Collagen to Calcium Phosphate Ratio of Formulation 5 
     Preparation of the Materials 
     Six bone graft substitute formulation prototypes were manufactured with pepsin-treated collagen and variable collagen to calcium phosphate granule ratios and dehydrothermal treatment (DHT) time, according to Table 5. The particle size range of calcium phosphate granules was between 500 μm and 1000 μm. Lyophilization was followed by DHT treatment in all samples. Lyophilization was performed according to the lyophilization program in Example 1, Table 2. DHT temperature was 110° C. for all formulations. All prototypes were gamma sterilized (25-30 kGy). After sterilization, each prototype was hydrated in preparation of handling evaluation. Hydration of prototypes was performed by placing each prototype in a petri dish and soaking with 5 cc of heparinized sheep blood. 
                                                                                         TABLE 5                   Prototype Formulations                        Collagen   Granule   Collagen               Prototype   Collagen   Conc   Final   Final   Slurry   Granule           ID   Composition   (mg/ml)   wt %   wt %   (ml)   (gr)   DHT                    Formulation   Pepsin   65   16   84   75   25   3       5-1   Treated                       days       Formulation   Pepsin   65   16   84   75   25   1       5-2   Treated                       days       Formulation   Pepsin   65   16   84   75   25   2       5-3   Treated                       hours       Formulation   Pepsin   65   12   88   64   36   3       5-4   Treated                       days       Formulation   Pepsin   65   12   88   64   36   1       5-5   Treated                       days       Formulation   Pepsin   65   12   88   64   36   2       5-6   Treated                       hours                    
Physical Handling of the Samples
 
     In order to determine the optimal ratio of collagen, granules, and DHT time for a sample, each prototype was evaluated based on its response to physical handling. Prototype samples were bended, twisted, pulled, and gently compressed to simulate handling conditions. Handling properties which were evaluated included compression resistance when wet, cohesiveness, moldability, flexibility, strength, and adhesiveness. 
     Handling properties of the prototypes were evaluated on a score from one (1) to five (5), with 1 being “poor” and 5 being “excellent.” Each prototype was evaluated four (4) times, each time by a different individual. 
     Results of Physical Handling 
     Results of the physical handling of each prototype are shown in Tables 6 and 7. 
     
       
         
               
             
               
               
               
               
               
               
             
           
               
                 TABLE 6 
               
             
             
               
                   
               
               
                 Handling Properties - Average Rating 
               
             
          
           
               
                   
                 Compression 
                   
                   
                   
                   
               
               
                 Prototype 
                 Resistance 
                   
                   
                 Flexi- 
                   
               
               
                 ID 
                 (Wet) 
                 Cohesiveness 
                 Moldability 
                 bility 
                 Strength 
               
               
                   
               
               
                 Formu- 
                 5 
                 5 
                 4 
                 5 
                 4 
               
               
                 lation 5-1 
                   
                   
                   
                   
                   
               
               
                 Formu- 
                 5 
                 5 
                 4 
                 4 
                 4 
               
               
                 lation 5-2 
                   
                   
                   
                   
                   
               
               
                 Formu- 
                 4 
                 5 
                 4 
                 4 
                 4 
               
               
                 lation 5-3 
                   
                   
                   
                   
                   
               
               
                 Formu- 
                 5 
                 4 
                 5 
                 5 
                 5 
               
               
                 lation 5-4 
                   
                   
                   
                   
                   
               
               
                 Formu- 
                 5 
                 5 
                 5 
                 5 
                 5 
               
               
                 lation 5-5 
                   
                   
                   
                   
                   
               
               
                 Formu- 
                 5 
                 5 
                 5 
                 5 
                 5 
               
               
                 lation 5-6 
               
               
                   
               
               
                 1 = poor; 2 = below average; 3 = average; 4 = above average; 5 = excellent 
               
             
          
         
       
     
     
       
         
               
             
               
               
             
           
               
                 TABLE 7 
               
             
             
               
                   
               
               
                 Results of Physical Handling 
               
             
          
           
               
                 Prototype 
                   
               
               
                 ID 
                 Comments 
               
               
                   
               
               
                 Formulation 
                 Dry: Minimal amount of flakes present in the vial. 
               
               
                 5-1 
                 Wet: Soaked up more than 90% of 5 cc blood (took about  
               
               
                   
                 8 to 10 minutes). Minimum granule loss when 
               
               
                   
                 manipulated. Compression resistant. Moldable, 
               
               
                   
                 flexible and strong when hydrated. 
               
               
                 Formulation 
                 Dry: Minimal amount of flakes present in the vial. 
               
               
                 5-2 
                 Wet: Soaked up more than 90% of 5 cc blood (took about 
               
               
                   
                 8 to 10 minutes). Minimum granule loss when  
               
               
                   
                 manipulated. Compression resistant. Moldable, 
               
               
                   
                 flexible and strong when hydrated. 
               
               
                 Formulation 
                 Dry: Minimal amount of flakes present in the vial. 
               
               
                 5-3 
                 Wet: Soaked up more than 90% of 5 cc blood (took about  
               
               
                   
                 8 to 10 minutes). Minimum granule loss when  
               
               
                   
                 manipulated. Compression resistance was slightly lower 
               
               
                   
                 than Formulations 5-1 and 5-2. Moldable, flexible and 
               
               
                   
                 strong when hydrated. 
               
               
                 Formulation 
                 Dry: Minimal amount of flakes present in the vial 
               
               
                 5-4 
                 Wet: Soaked up more than 90% of 5 cc blood (took about  
               
               
                   
                 6 to 8 minutes, faster than Formulations 5-1, 5-2 and 
               
               
                   
                 5-3). Minimum granule loss when manipulated. 
               
               
                   
                 Compression resistant. After manipulating, bounces back  
               
               
                   
                 to its original shape (good shape memory property).  
               
               
                   
                 The best performer (same as Formulation 5-5) 
               
               
                 Formulation 
                 Dry: Minimal amount of flakes present in the vial 
               
               
                 5-5 
                 Wet: Soaked up more than 90% of 5 cc blood (took about  
               
               
                   
                 6 to 8 minutes, faster than #1, 2, and 3 prototypes). 
               
               
                   
                 Minimum granule loss when manipulated. Compression 
               
               
                   
                 resistant. After manipulating, bounces back to its 
               
               
                   
                 original shape (good shape memory property). The  
               
               
                   
                 best performer (same as Formulation 5-4) 
               
               
                 Formulation 
                 Dry: Minimal amount of flakes present in the vial 
               
               
                 5-6 
                 Wet: Soaked up more than 90% of 5 cc blood. Minimum  
               
               
                   
                 granule loss when manipulated. Compression resistant. 
               
               
                   
                 After manipulating, bounces back to its original shape 
               
               
                   
                 (good shape memory property). Handling performance  
               
               
                   
                 was slightly lower than Formulations 5-4 and 5-5 but still  
               
               
                   
                 showed excellent handling characteristics. 
               
               
                   
               
             
          
         
       
     
     All prototypes performed well and demonstrated acceptable handling properties, but some prototypes performed better than others. Formulation 5-4 and Formulation 5-5 prototypes performed the best as a compression resistant matrix. While the following prototypes each had acceptable handling properties, their level of handling performance decreased in the following order: Formulation 5-6, Formulation 5-1, Formulation 5-2, Formulation 5-3. Results also indicated that prototypes having more granules resulted in a stiffer scaffold. Blood uptake of Formulation 5-4, 5-5 and 5-6 were faster than Formulation 5-1, 5-2 and 5-3. This may be attributed to higher granule content in Formulations 5-4, 5-5 and 5-6. 
     Discussion &amp; Conclusions 
     According to the results, all prototypes showed acceptable handling characteristics, but Formulations 5-4 and 5-5 performed most optimally. Formulations 5-4, 5-5 and 5-6 were compression resistant. Whereas Formulations 5-4 and 5-5 had the most optimal handling characteristics, Formulation 5-6 also performed well and has significant manufacturing benefits as a result of its short DHT time. 
     EXAMPLE 3 
     Collagen Preparation 
     Four bone graft substitute prototypes were manufactured using 4 different calcium phosphate (CaP) granule lots, according to Table 8. Properties of each CaP granule lot are shown in Table 9. During manufacture, each sample was prepared using fresh collagen dough, and granules were mixed with collagen using a new blending technique. 
     The prototype samples were manufactured by using a new blending technique as follows. Low concentration collagen (LCC) and high concentration collagen (HCC) were prepared. Type I bovine collagen from tendon source treated with 0.05% pepsin was suspended in 10% isopropyl alcohol and mixed with 0.1N HCl. The soluble collagen was adjusted to the low (12.5 mg/g) and high (100 mg/g) concentration by controlling the amount of 10% isopropyl alcohol and HCl solution. The LCC was mixed with the calcium phosphate granules by hand and was left to sit for 30 minutes. The LCC/CaP granule slurry was then blended with the HCC by mixer for nine minutes. The final concentration of the collagen solution was 65 mg/g. At the end of the blending process, the collagen/granule mixture was poured into molds. Thus, each prototype has a composition as shown in Table 10. 
     The same lyophilization program was performed for each sample according to the lyophilization program in Example 1, Table 2. Lyophilization was followed by Dehydrothermal (DHT) treatment in all samples. DHT temperature was 110° C. for all samples. All prototypes were gamma sterilized (25-30 kGy). Each prototype underwent XRD analysis, SEM analysis and qualitative handling evaluation analysis to determine the effect of the new blending technique on granule phase composition and porosity. 
     
       
         
               
             
               
               
               
               
             
           
               
                 TABLE 8 
               
             
             
               
                   
               
               
                 Bone Graft Substitute Prototypes 
               
             
          
           
               
                   
                   
                 Calcium 
                   
               
               
                   
                   
                 Phosphate 
                 Dimensions 
               
               
                   
                 Prototype ID 
                 Granule Lot # 
                 (L × W × H) 
               
               
                   
               
               
                   
                 Formulation A 
                 1 
                 50 × 10.5 × 12 mm  
               
               
                   
                   
                   
                 (±1 × ±1 × ±1 mm) 
               
               
                   
                 Formulation B 
                 2 
                 50 × 10.5 × 12 mm 
               
               
                   
                   
                   
                 (±1 × ±1 × ±1 mm) 
               
               
                   
                 Formulation C 
                 3 
                 50 × 10.5 × 12 mm 
               
               
                   
                   
                   
                 (±1 × ±1 × ±1 mm) 
               
               
                   
                 Formulation D 
                 4 
                 50 × 10.5 × 12 mm 
               
               
                   
                   
                   
                 (±1 × ±1 × ±1 mm) 
               
               
                   
               
             
          
         
       
     
     
       
         
               
             
               
               
               
               
             
               
               
               
               
             
           
               
                 TABLE 9 
               
             
             
               
                   
               
               
                 Characteristics of CaP Granules 
               
             
          
           
               
                 CaP Granule 
                   
                   
                   
               
               
                 Lot # 
                 Granule Size 
                 TCP % 
                 Porosity % 
               
               
                   
               
             
          
           
               
                 1 
                 500-1000 μm 
                 94 
                 75 
               
               
                 2 
                 500-1000 μm 
                 100 
                 75.6 
               
               
                 3 
                 500-1000 μm 
                 100 
                 68 
               
               
                 4 
                 500-1000 μm 
                 93 
                 68 
               
               
                   
               
             
          
         
       
     
                                       TABLE 10                   Prototype Formulation            Final Conc. (mg/g) of   Collagen Final   Granule Final   DHT       Collagen Solution   wt %   wt %   Duration               65   12   88   24 hr                    
3.1 X-Ray Diffraction (XRD) Analysis
 
     3 samples of each prototype underwent XRD analysis and results are summarized in Table 6. All samples were within specification, TCP&gt;90% and HA&lt;10%. The combination of collagen with granules did not influence the granule composition. 
                                                                             TABLE 11                   XRD Analysis Results                            Other   Ca/P           Sample ID   β TCP %   HA %   phases   ratio                        Formulation A   1   94   6   No   1.51               2   94   6   No   1.51               3   94   6   No   1.51           Formulation B   1   96   4   No   1.51               2   94   6   No   1.51               3   95   5   No   1.51           Formulation C   1   100   0   No   1.50               2   100   0   No   1.50               3   100   0   No   1.50                    
3.2 Scanning Electron Micrograph (SEM) Analysis
 
     Prior to SEM evaluation, 2 samples of each prototype were divided into smaller pieces and samples were fixed on a SEM sample stub and coated with gold-palladium to increase the conductivity of the samples. Per SEM evaluation, it is evident that surface morphology of granules did not change after mixing with collagen. 
     Qualitative Handling Evaluation 
     Each prototype sample was hydrated with 6 cc of heparinized sheep blood. Prior to handling evaluation, samples were kept in blood until most of the blood was absorbed by the sample. 3 samples of each prototype were evaluated by a total of 5 expert users. All of the evaluators agreed that the speed of blood uptake was very fast for all samples and, overall, all of the samples showed excellent handling properties. Handling characteristics are summarized in Table 12. 
     
       
         
               
             
               
               
             
           
               
                 TABLE 12 
               
             
             
               
                   
               
               
                 Qualitative Handling Evaluation 
               
             
          
           
               
                 Prototype ID 
                 Comments 
               
               
                   
               
               
                 Formulation  
                 Dry: Minimal amount of flakes present in the vial. 
               
               
                 A 
                 Wet: Soaked up more than 95% of 6 cc blood within  
               
               
                   
                 90 seconds. Minimum granule loss when manipulated. 
               
               
                   
                 Compression resistant. After manipulating, bounces 
               
               
                   
                 back to its original shape. After longer 
               
               
                   
                 manipulation, forms a cohesive and a workable putty.  
               
               
                   
                 Minimal granule loss in putty form. 
               
               
                 Formulation  
                 Dry: Minimal amount of flakes present in the vial. 
               
               
                 B 
                 Wet: Soaked up more than 95% of 6 cc blood within  
               
               
                   
                 90 seconds. Minimum granule loss when manipulated.  
               
               
                   
                 Compression resistant. After manipulating, bounces  
               
               
                   
                 back to its original shape. After longer manipulation,  
               
               
                   
                 forms a cohesive and a workable putty. Minimal  
               
               
                   
                 granule loss in putty form. 
               
               
                 Formulation  
                 Dry: Minimal amount of flakes present in the vial. 
               
               
                 C 
                 Wet: Soaked up more than 95% of 6 cc blood within  
               
               
                   
                 90 seconds. Minimum granule loss when manipulated. 
               
               
                   
                 Compression resistant. After manipulating, bounces 
               
               
                   
                 back to its original shape. After longer manipulation, 
               
               
                   
                 forms a cohesive and a workable putty. Minimal granule 
               
               
                   
                 loss in putty form. 
               
               
                   
               
             
          
         
       
     
     The blending technique described herein allows for the mixing of granules and collagen without any phase separation. Per XRD and SEM results, mixing the granules using the new technique results in no change in microstructure or phase composition of the granules. According to the qualitative handling evaluation, all samples soaked up most of the blood within 90 seconds. When the prototypes were manipulated, minimum granule loss was observed. All samples were compression resistant, and after compressing on each sample, all bounced back to their original shape. Additionally, when the samples were firmly compressed and manipulated by kneading and tearing, all samples showed a putty type of consistency. This formed putty was cohesive and when handled showed minimum granule loss, which is a desired property for a putty.