Association of vancomycin and calcium phosphate by dynamic compaction: in vitro characterization and microbiological activity

Biomaterials 22 (2001) 2481}2487

Association of vancomycin and calcium phosphate by dynamic compaction: in vitro characterization and microbiological activity H. Gautier 
*, G. Daculsi , C. Merle 
Centre de recherche sur les mate& riaux d+inte& reL t biologique, Equipe INSERM 99-03; Faculte& de chirurgie dentaire, 1, place Alexis Ricordeau, 44042 Nantes Cedex, France
Laboratoire de Pharmacie Gale& nique, Faculte& de Pharmacie, 1, rue Gaston Veil, 44035 Nantes Cedex, France Received 24 July 2000; accepted 7 December 2000

Abstract Dynamic compaction has rarely been used to produce drug-delivery devices in granule form. This report considered four processes associating vancomycin and compared dynamic compaction with wet granulation, a classical method. In the wet granulation study, vancomycin was associated with biphasic calcium-phosphate (BCP) granules either by adsorption or incorporation with a new granulation. In the dynamic compaction study, BCP powder was compacted at 1.1, 1.5 and 1.9 MPa. The compacts obtained were crushed and sieved (200}500
m), and the vancomycin solution was adsorbed on the resulting granules. After crushing and sieving, the compaction of BCP and vancomycin powders produced vancomycin-loaded granules. In each study, 4.76% of vancomycin was associated with BCP. Granules were characterized in terms of porosity, vancomycin release and vancomycin biological activity. Physicochemical studies of BCP and vancomycin showed their structural integrity after dynamic compaction, which prolonged vancomycin release time from 1 to 6 days. However, a microbiological assay indicated that vancomycin had been altered since only 27.7% was found to be active.  2001 Elsevier Science Ltd. All rights reserved. Keywords: Vancomycin; Biphasic calcium phosphate; Drug-delivery system; Dynamic compaction; Wet granulation; Ceramic

1. Introduction In the event of severe osteoarticular infection and the risk of osseous necrosis and vascular thrombosis, surgical treatment is required to eliminate macroscopic infected tissues [1]. Antibiotics are administered systemically at the excision site in su$cient concentrations to eradicate the infection. An alternative to systemic treatment involves the implantation of a biomaterial that can release antibiotics in situ for prophylactic and curative purposes. Antibiotics penetrate bone easily, and their sustained release prevents proliferation of germs from the surgical operation. Numerous bone substitute biomaterials such as cements and calcium-phosphate products are used, and many antibiotics can be associated with these materials, including gentamycin [2}4] and vancomycin [5}8].

* Correspondence address: Laboratoire de Pharmacie GaleH nique, FaculteH de Pharmacie, 1, rue Gaston Veil, 44035 Nantes Cedex, France. Fax: #33-02-4041-2877. E-mail address: [email protected] (H. Gautier).

Ceramics, which are frequently used as bone substitutes [9] and for the delivery of matrix therapeutic agents [10], consist of variable proportions of hydroxyapatite and tricalcium phosphate-
. Their intermediary degradation characteristics [9], which allow gradual resorption at the same time as osseous colonization, provide for total bone substitution. Biphasic calcium phosphate (BCP), the material used in the present study, has proved e$cient for this purpose [11}13]. Vancomycin appears to be the most suitable antibiotic for administration in terms of prophylaxis, e$ciency and non-toxicity. It is also e!ective in treating osteomyelitis and preventing osseous staphylococcal infections [14,15]. Di!erent processes of therapeutic agent}matrix association can be used, such as mixing [16}19], centrifugation [20}22] or adsorption [23,24], to facilitate contacts between the biomaterial and the therapeutic agent or to achieve compaction. The compaction of melted powders allows the preparation of sustained release forms, either by isostatic compression [25,26] or dynamic compaction. The latter process is based on powder consolidation by projectile impact (University of Nantes, patent no. 9212837). This technique is suitable for the production of

0142-9612/01/$ - see front matter  2001 Elsevier Science Ltd. All rights reserved. PII: S 0 1 4 2 - 9 6 1 2 ( 0 0 ) 0 0 4 3 6 - 1

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bulk material from powders and has been used to create blocks of biomaterial loaded with human growth hormone [27] and vancomycin [28]. It was used in the present study to prepare BCP granules loaded with vancomycin. A particle size of 200}500
m was used to promote bone growth and down-modulation of in#ammatory processes. The aim of this study was to evaluate vancomycin-loaded BCP granules prepared by dynamic compaction and to compare them with granules obtained by a more classical process: wet granulation. These granules were characterized for their structure, porosity, vancomycin release and vancomycin microbiological activity.

were mechanically sieved for 10 min to collect the 200}500
m fraction. One milliliter of a vancomycin solution in distilled water (12.5 mg/ml) was deposited at the surface of 250 mg of sieved BCP granules and placed in an oven at 373C until complete evaporation of the solution. B granules. Sixty grams of sintered granules prepared by wet granulation were again subjected to wet granulation using a vancomycin solution (115 mg/ml of distilled water). The mixture was granulated in an oscillated granulator, and the vancomycin-BCP granules obtained were then oven-dried at 373C to a constant weight. Sieving was performed for 10 min to recover the 200}500
m fraction.

2. Materials and methods

2.2.3. Preparation of BCP}vancomycin by dynamic compaction A preliminary study [30] showed that BCP granules obtained by wet granulation had a better compactibility than BCP powder. C granules. Dynamic compaction was performed using compacting equipment (Centre de recherche sur les mateH riaux d'inteH re( t biologique, Nantes, France). Four grams of BCP granules obtained by wet granulation were compressed at 1 ton in a compaction chamber with a press (Power Team 25 ton, SPX Corporation, USA). The chamber was then introduced into the compaction compass, and the powder was compacted at 1.1, 1.5 or 1.9 MPa. The compacts obtained were crushed in a grooved roller breaker (TG2S, Erweka Apparatebau GmbH, Germany) and sieved for 10 min using a RotoLab sieve (Chauvin, France) to collect the 200}500
m fraction. One milliliter of a vancomycin solution (12.5 mg/ml of distilled water) was deposited on the surface of 250 mg of each di!erent compressed batch of BCP granules. The wet masses were then placed in a 373C oven until complete evaporation of the solvent. The three batches of vancomycin-associated BCP granules were referred to, respectively, as C1, C2 and C3 for an applied dynamic compaction of 1.1, 1.5 and 1.9 MPa. D granules. Twenty grams of BCP were blended with 1 g of vancomycin powder (Turbula T2C, WAB, Switzerland) for 10 min. Four grams of this batch were precompacted and then compacted at 1.1, 1.5 or 1.9 MPa. The resulting compacts were crushed and sieved into a 200}500
m powder. The three batches of granules prepared by direct incorporation of vancomycin into BCP were referred to, respectively, as D1, D2 and D3 for an applied dynamic compaction at 1.1, 1.5 and 1.9 MPa.

2.1. Materials and reagents Vancomycin hydrochloride (both Vancocine威 for oral administration and the clinical laboratory standard) was obtained from Lilly France (Saint Cloud, France), and FTIR grade potassium bromide from Sigma-Aldrich (St. Quentin Fallavier, France). Six-well culture plates were supplied by AES (Combourg, France), and Millicell威 culture chambers (CM PICM) from Millipore (St. Quentin, France). AM2 Difco antibiotic medium was supplied by Fisher (Elancourt, France), and brain heart broth by Biokar Diagnostics (Beauvais, France). Micrococcus luteus (ATCC 9341) was used for the microbiological test. 2.2. Preparation of vancomycin-loaded BCP granules 2.2.1. Preparation of BCP powder Apatite powder was synthesized in the laboratory using the hydrolysis reaction protocol previously described [29], puri"ed by washing, and then oven-dried at 403C to a constant weight. A heat treatment was applied to the powder in a Vecstar furnace (Eurotherm, Switzerland) to transform the calcium-de"cient apatite into BCP. The sintering protocol consisted of heating at 303C/min to 10503C, holding for 5 h at 10503C, and slow cooling of samples (33C/min to 203C) to avoid heat shock. Vancomycin was associated with BCP by di!erent means, but always in a 4.76% proportion (w/w). 2.2.2. Preparation of BCP}vancomycin granules by wet granulation A granules. Apatite powder was agglomerated by wet granulation using distilled water in a planetary blender (Braun France). The mass obtained was forced through the mesh of two grids (1.040 and 0.630 mm) in an oscillated granulator (Erweka AR 400 Apparatebau GmbH, Germany). The granules obtained were oven-dried at 373C to a constant weight, and sintering was applied as previously described. Thirty grams of sintered granules

2.3. Characterization of BCP}vancomycin granules 2.3.1. Physicochemical analysis 2.3.1.1. X-ray diwraction and infrared spectroscopy. X-ray di!raction pro"les and Fourier transform infrared

H. Gautier et al. / Biomaterials 22 (2001) 2481}2487

(FTIR) spectroscopy of BCP powder before and after dynamic compaction at 1.9 MPa were performed, respectively, on a PW 1730 X-ray generator (Philips, France) and a magna 500 FTIR spectrometer (Nicolet Inc., Paris, France) to investigate the chemical and crystallographic features of the material. 2.3.1.2. Nuclear magnetic resonance. H and C-NMR analyses were performed on a D O solution of van comycin prior to and after dynamic compaction at 1.9 MPa on a Bruker Avance DRX 500 MHz spectrometer (Bruker, France). 2.3.2. Granule porosity A granule porosity study was performed by scanning electron microscopy (SEM) (JSM-6300, Jeol, Japan) on granules prepared without vancomycin and dehydrated in alcohol and acetone. After inclusion in glycolmethacrylate (48 h), the blocks obtained were polished and their porosity determined by SEM coupled to a semi-automatic image analyzer (Quantimet 500, Leica, UK) after gold-palladium coating (Emscope AEI230, Ashford, UK). Four granules from each batch were analyzed. Results are expressed as the percentage of pore surface area. Porosity assessments were analyzed using Student's t-test (0.05 level) relative to granules formulated by dynamic compaction at the three pressures and those obtained by wet granulation. 2.4. Vancomycin release proxles Vancomycin release pro"les were assessed on each type of BCP}vancomycin granule (A}D), using a previously described dissolution test [24]. Two hundred and "fty milligrams of granules were deposited in a Millicell culture chamber equipped with a Biopore威 membrane. The chambers were immersed in six-well culture plates containing 13 ml of distilled water that were subsequently placed in an oven at 373C on a 3D rocking platform (Stuart Scienti"c, STR 9, UK) (5 rpm). Aqueous solutions were removed and replaced with fresh distilled water at 1, 2, 3, 4, 6, 24 and 48 h and then day to day. The amount of vancomycin released in aqueous solutions was determined by an UV-visible spectrophotometer assay (Shimadzu UV-1605 UV Visible spectrophotometer, Roucaire, France) at 280 nm. All experiments were performed in triplicate. Results are expressed as the percentage of vancomycin released $SD over time. Samples were frozen at !803C for the microbiological assay. For both processes of BCP}vancomycin granule formulation, vancomycin release pro"les over time were studied statistically by means of SYSTAT software [31] using two-way analysis of variance followed by a pairwise mean multiple comparison Tukey test.

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2.5. Vancomycin microbiological activity A microbiological assay was performed to estimate the active amount of vancomycin released during the in vitro dissolution test. This assay consists in measuring the inhibition diameter of a sensitive bacterium by an antibiotic. Micrococcus luteus (ATCC 9341) grown overnight on trypticase soy broth was suspended in brain heart broth and incubated at 373C. After 24 h, 4.5 ml of the Micrococcus suspension at 10 bacteria per milliliter were incorporated into 180 ml of agar AM 2 heated at 453C. The agar was poured into a sterile glass plate and left for 2 h at room temperature. At each assay, a new standard curve was prepared with vancomycin (clinical laboratory standard) at concentrations of 2, 4, 8, 16, 32, 64 and 128
g/ml. One hundred and twenty microliters of samples and standards diluted in sterile water were deposited in triplicate in calibrated holes made in the agar. Standards and samples were randomly located in di!erent areas of the plate, which was left for 2 h at room temperature and then incubated for 24 h at 373C before measurement of inhibition diameters. The results for inhibition diameters were converted in terms of concentration (
g/ml) according to the standard curve. All experiments were performed in triplicate. Results are expressed as the mean$SD as a function of time. Control samples containing only dynamically compacted BCP were also measured by the microbiological method. The results for vancomycin spectrophotometric and microbiological concentrations were compared in order to determine the percentage of active vancomycin. 3. Results 3.1. Characterization of BCP}vancomycin granules 3.1.1. Physicochemical analysis 3.1.1.1. X-ray diwraction and infrared spectroscopy. Infrared and X-ray di!raction pro"les of BCP powders before and after dynamic compaction at 1.9 MPa showed no changes in BCP powder during compaction [32}33]. The HA and
-TCP phases of BCP were clearly identi"ed. The HA/
-TCP ratio of BCP powders before and after dynamic compaction was, respectively, 42.2/57.8 and 40.9/59.1, corresponding to a calcium-phosphate ratio of 1.60. 3.1.1.2. Nuclear magnetic resonance. Spectral data for vancomycin powders showed that typical vancomycin peaks were recovered after dynamic compaction [34]. 3.1.2. Granule porosity The porosity percentages of granules prepared by wet granulation and dynamic compaction are shown in

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Table 1 Porosity percentages obtained by SEM and image analysis for granules prepared by wet granulation and dynamic compaction at 1.1, 1.5 and 1.9 MPa Granule formulation

Porosity (%$SD)

Wet granulation Dynamic compaction 1.1 MPa 1.5 MPa 1.9 MPa

37.7$6.8 12.4$3.6 12.3$4.0 9.9$4.7

Porosity percentages are expressed as the mean$SD.

Table 1. Porosity ranged from 37.7%$6.8 (A granules) to 9.9%$4.7 (C granules). Statistical analysis (t-test at the 0.05 level) showed no signi"cant di!erences between the porosity of granules obtained at 1.1, 1.5 and 1.9 MPa, whereas a signi"cant di!erence was found between wet granulation granules and dynamic compaction granules obtained at 1.1, 1.5 and 1.9 MPa ( p(10\ for all granules).

for granules C and D prepared by dynamic compaction, but only within 3 days for granules A and B prepared by wet granulation. A lag time of approximately 6 h was observed for all release pro"les. Beyond this period, vancomycin was released until complete delivery. Seventy-"ve percent of the vancomycin associated with BCP by dynamic compaction was released between 38.7 h for C3 granules and 126.2 h for C2 granules and between 40.8 h for D3 granules and 57.9 h for D1 and D2 granules. For granules prepared by wet granulation, 75% of the vancomycin was released between 11.1 h (A granules) and 21.9 h (B granules). Statistical comparison of granule vancomycin release pro"les showed no signi"cant di!erences (p"0.36) for vancomycin adsorbed or directly incorporated into granules, regardless of the dynamic compaction formulation technique used. However, vancomycin release was significantly faster from granules prepared by wet granulation than from those prepared by dynamic compaction ( p(10\ for vancomycin adsorbed on granules and p"10\ for vancomycin directly incorporated into the granule mass). Moreover, vancomycin release was not signi"cantly modi"ed by compaction force.

3.2. Vancomycin release proxles 3.3. Vancomycin microbiological activity The amounts of vancomycin released from A to D granules over time were determined from the results of in vitro dissolution tests (Fig. 1). The pro"les showed that vancomycin was released within approximately 4}6 days

The concentrations obtained with control samples containing only dynamically compacted BCP granules were 0.000.

Fig. 1. Release pro"les for vancomycin from BCP A (a), B (b), C (c) and D (d) granules.

H. Gautier et al. / Biomaterials 22 (2001) 2481}2487 Table 2 Vancomycin concentrations measured by UV spectrophotometry and microbiological assay, and the percentage of microbiological active vancomycin released from granules Concentrations (mg/ml$SD) UV assay

Microbiological assay

0.33$0.06

0.09$0.03

Active vancomycin (%)

27.7

Experimental results obtained with UV spectrophotometric and microbiological measurements on 24 h samples allowed evaluation, respectively, of the vancomycin concentrations and the percentage of active vancomycin released from granules. The results in Table 2 indicate that the percentage of active vancomycin released from granules prepared by dynamic compaction was 27.7%.

4. Discussion Calcium-phosphate blocks or granules are often used as bone substitutes in the prophylaxis and treatment of osseous infections. Granules are more convenient than blocks and allow the replacement of a large bone volume. As blocks are di$cult to "t into cavities, vacant areas are often observed between block and bone. Granules can be prepared by classical techniques such as wet granulation, which is frequently used in the pharmaceutical industry to prepare pellets. However, block formation techniques followed by a granulation procedure can also be used. This study used a recently developed powder compaction technique to consolidate calcium-phosphate powders. In this process, particle surfaces are highly deformed, producing interparticulate bonding in a onestep process. This occurs during the passage of a shock wave through the powder. As this technique allows the compact to be formed without a sintering step, a heatsensitive therapeutic agent can be associated with a calcium-phosphate powder without denaturing the active element. The agent and powder can be associated before compaction, provided that the energy of the shock does not denature the agent. Previous studies showed the advantage of using dynamic compaction to obtain compacts of calcium-phosphate biomaterials [35] and associate therapeutic agents with those materials [27,28]. These studies investigated the association of growth factor and glycopeptidic antibiotic with BCP. Human growth hormone (hGH) and vancomycin were associated with BCP and then compacted. They were then extracted from compacts and assayed, respectively, by monoclonal antibodies plus SDS-PAGE and by immunoenzymology. The results showed that neither hGH nor vancomycin was dena-

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tured in terms of structure and biochemical characteristics [28]. In a complementary study concerning hGH alone [27], an MTT-eluted stain bioassay showed that the biological activity of hGH was completely preserved after dynamic compaction. The present study characterized two processes associating vancomycin with BCP to obtain granules, i.e. wet granulation and wet granulation followed by dynamic compaction. As pressures of 0.5, 0.7 and 0.9 MPa had been previously tested and found insu$cient to produce BCP compaction, higher pressures of 1.1, 1.5 and 1.9 MPa were used. The granules obtained were analyzed and compared in terms of physicochemistry, porosity, vancomycin release and vancomycin microbiological activity. Physicochemical characterization of calcium-phosphate granules by X-ray di!raction, infrared spectroscopy and nuclear magnetic resonance showed that the structures of BCP and vancomycin were unchanged by dynamic compaction at 1.9 MPa. This is concordant with the results of a previous study [35] showing that the structures of powders such as hydroxyapatite,
-calcium phosphate and octacalcium phosphate were conserved after 2 MPa dynamic compaction. A previous study also showed that BCP powder structure was unchanged after dynamic compaction [28]. SEM studies showed that granule porosity depended on the manufacturing process, ranging between 37.7$ 6.8 and 9.9$4.7%. Granule porosity with dynamic compaction was three- to four-fold lower than with wet granulation. In fact, the wet granulation process is performed during a single step in which densi"cation occurs, whereas granule preparation is done in two steps with the dynamic compaction process: densi"cation by powder volume reduction (which gives a compact with lower density) followed by crushing. This volume reduction is correlated with the pressure applied (porosity is reduced when compaction pressure is high). As bone ingrowth is correlated with material porosity [36], the choice of preparation process allows various granules to be obtained. Vancomycin release pro"les showed that delivery lasted for several days and that variations depended on how the therapeutic agent was incorporated into the biomaterial. Wet granulation (A and B granules), based on particle association by powder humectation [37], gave lower release than when granules were prepared by closer association during compaction (C and D granules). The lag time of 6 h observed in each release pro"le probably corresponded to the time needed to impregnate the granules and allow release of the therapeutic agent. Wet granulation, the process allowing faster delivery, released the associated vancomycin in a maximum of 3 days. When vancomycin was associated by direct incorporation into BCP granules (B granules), the release time was three times as long as when the drug was adsorbed

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on BCP granules (A granules). Dynamic compaction increases the period of vancomycin release to 4}6 days. Granules obtained by this process form a matrix that releases the therapeutic agent slowly, depending on the binding force (not yet determined) of vancomycin to calcium phosphate. The pressure of dynamic compaction had no signi"cant in#uence on release time, and there was no signi"cant di!erence for vancomycin adsorbed on granules prepared by dynamic compaction (C granules) or compacted with BCP granules (D granules). In the "rst case, the water of the dissolution medium penetrated into pores to release the therapeutic agent, and in the second case a vancomycin dissolution}di!usion process worked from the periphery toward the center of the granule. The absence of any di!erence in the dissolution rate can be explained by the low particulate size of the granules (200}500
m). The results obtained con"rm the notion of an increase in the release time of vancomycin closely associated with BCP powder, i.e. compacted with BCP. These results are in agreement with those previously obtained by us for isostatic compression, which allowed a three- to "ve-fold increase in release time compared to wet granulation [25]. A microbiological assay was used to determine vancomycin released biological activity. Although standard documents recommended others test-microorganisms (Bacillus subtilis or cereus) Micrococcus luteus was chosen because of its good sensibility to vancomycin (large and reproductible inhibition diameters). The aim of this experiment was not to test the bactericidal kinetic of vancomycin against Micrococcus luteus but to assay the quantity of active vancomycin. A comparison of the amounts of activity detected by the spectrophotometric assay and the microbiological assay showed that only 28% of the released vancomycin was active. This compaction technique is known to cause large but brief local temperature increases in compacts, which are not quanti"able but high enough to induce grain joint formation [38]. This could denature vancomycin which yet remains stable for 6 h at 803C. Though a previous study [28] indicated that vancomycin was not denatured by dynamic compaction, the assay used was immunoenzymatic (EMIT) and thus only quantitative. Other processes not involving high temperatures, such as isostatic compression, have not denatured vancomycin associated with BCP [26]. BCP granules showed no antibacterial e!ects, in accordance with a previous study by our group [26]. However, Opalchenova et al. [39] and Ingram et al. [40] noted such e!ects, probably because of di!erences in their experimental conditions.

5. Conclusion The processes used to prepare implantable bone materials are known to have an in#uence on the release of

therapeutic agents. In addition to the techniques classically used to prepare drug delivery devices, new processes such as dynamic compaction, that closely associate the therapeutic agent with a calcium-phosphate matrix, are now available. Two processes (wet granulation and dynamic compaction) and two means of incorporating the therapeutic agent (adsorption and direct incorporation) were studied here. These procedures allowed four types of granules to be obtained, which were characterized by variable porosity and sustained release. This study showed that dynamic compaction of vancomycin and BCP powder produces vancomycin-loaded granules with low porosity and that dynamic compaction increases the release time of vancomycin in the medium two- to sixfold compared to wet granulation. A 6-day period of vancomycin release from granules prepared by direct incorporation of the therapeutic agent into BCP by dynamic compaction seems suitable for treatment or prophylaxis of bacterial proliferation during a surgical operation. However, dynamic compaction reduces the microbiological activity of vancomycin.

Acknowledgements The authors are grateful to Prof. P. Richomme from the SCRMN, University of Angers, for NMR analysis; Prof. Y. Pegon from the analytical chemical laboratory, University of Nantes, for lending a UV spectrophotometer; Dr. J. Caillon for assistance with the microbiological assay; and Mrs. Miegeville for performing microbiological analysis. The authors are also grateful to Mr. C. Boiteux for performing the dynamic compaction and to Mr. J. Gray for English correction.

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