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Table of Contents   
ORIGINAL RESEARCH  
Year : 2020  |  Volume : 31  |  Issue : 3  |  Page : 481-485
Hydroxyapatite carriers as drug eluting agents—An In Vitro analysis


1 Department of Oral & Maxillofacial Surgery, Government Dental College, Thiruvananthapuram, Kerala, India
2 Bioceramics Laborotary, SCTIMST, Thiruvananthapuram, Kerala, India

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Date of Submission23-Apr-2019
Date of Acceptance02-Oct-2019
Date of Web Publication06-Aug-2020
 

   Abstract 


Introduction: Hydroxyapatite based drug carriers offer a customized alternative to the delivery of pharmacologic agents in the osseous skeleton. They have an added advantage of being biocompatible and osteoconductive. This in vitro study aims to quantify the drug eluting properties of HA granules by spectrophotometry. Materials and Methods: HA and HASi beads were loaded with gentamicin/ amoxycillin- clavulanate/ vancomycin and grouped into 5. Drug elution was evaluated by means of UV spectrophotometry. Results: Drug eluent levels were well above bactericidal levels in all 5 groups. Conclusion: HA and HASi are viable options for clinicians for targeted drug delivery.

Keywords: Drug carriers, drug eluting agents, hydroxyapatite

How to cite this article:
Simon D, Kumar K A, Sivadasan SB, Varma H, Balan A. Hydroxyapatite carriers as drug eluting agents—An In Vitro analysis. Indian J Dent Res 2020;31:481-5

How to cite this URL:
Simon D, Kumar K A, Sivadasan SB, Varma H, Balan A. Hydroxyapatite carriers as drug eluting agents—An In Vitro analysis. Indian J Dent Res [serial online] 2020 [cited 2020 Sep 18];31:481-5. Available from: http://www.ijdr.in/text.asp?2020/31/3/481/291483



   Introduction Top


Bone is a highly complex and evolved composite connective tissue, comprising of organic and inorganic components arranged in a distinct fashion.[1] It has a mineralized extracellular matrix made up of collagen and reinforced with mineral, mainly apatite which accounts for approximately 69% of the weight of fresh bone. It is affected by infection, malignancy, congenital, and autoimmune diseases as well as consequences related to aging like osteoporosis. The WHO has designated the present and immediate past decade as the “Bone and joint decade,” to emphasize the enormity of the problem.[2]

Therapeutic intervention helps to restore and regenerate the diseased tissues. Bisphosphonates, hormones, monoclonal antibodies, selective estrogen receptor modulators (SERMs), chemotherapeutic agents, analgesics, growth factors, and antibiotics are some of the agents used systemically, in the treatment of bony pathology. Drugs administered systemically are first absorbed into the circulation and distributed throughout the body and may penetrate the bone poorly. Tissue engineering has developed drug delivery systems (DDS) and controlled drug release systems (CDRS) that specifically target the bone, enhancing therapeutic efficacy and minimizing systemic toxicity.

Synthetic hydroxyapatite (HAp) [(Ca10(PO4)6(OH)2] is the workhorse of these systems, due to its unique properties. These materials are available as injectable cements, granules, or macroporous blocks.[3] The drug carrying and eluting capacity of these carriers needs careful evaluation and calibration before clinical application. Simon et al. have conducted anin vitro study comparing the release of gentamicin from commercially available PMMA (Polymethyl methacrylate) beads and indigenously manufactured nanoporous HAp granules.[4] The drug eluting characteristics of gentamicin bone cements and indigenously designed nanoporous bioceramic granules were analyzed and compared spectrophotometrically, and yielded drug concentration >100 μg/g from both samples. This study aims to compare the drug loading and elution of gentamicin, vancomycin, and amoxycyllin clavulanate from nanoporous HA and HASi granules, in an attempt to authenticate the drug carrying and release characteristics of these synthetic bioceramic implants.


   Materials and Methods Top


Antibiotic loading

Nanoporous hydroxyapaptite granules [Figure 1] and bioactive glass-coated (HASi) beads [Figure 2] were prepared by an in-house developed method (Bioceramic Laboratory, Biomedical Technology Wing, SCTIMST, Trivandrum). Biscuit fired HA beads were soaked in silica solution and sintered. HA and HASi nanoporous beads of average diameter 30+/- 1 mm were autoclaved and used for antibiotic impregnation. Three antibiotics, namely gentamicin sulfate (Genticyn, 80 mg/2 ml, Abbott), amoxicillin and potassium clavulanate injection IP 1.2 g, amoxicillin content 1,000 mg, Glaxo Smith Kline), and vancomycin hydrochloride (Vanild 500 mg, Cipla) were used in the study. Ethical committee clearance was obtained on 23/7/2013.
Figure 1: SEM image of nanoporous HA granule

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Figure 2: SEM image of bioactive glass -coated (HASi ) bead

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For preparing amoxicillin and potassium clavulanate solution, 1.2 g powder was dispersed in 20 ml sterile water; for preparing vancomycin solution 500 mg powder was dispersed in 10 ml sterile water and gentamicin sulfate solution was used as such. Antibiotics were loaded by vacuum loading with a help of a syringe assembly. One gram weight of the beads was put in the syringe barrel, and 5 ml of the drug solution was added, the syringe plunger was placed into correct position and on pulling the plunger a vacuum was created. The beads were held under the vacuum in the drug solution for 2 min so that the pores and pore channels of the beads got filled with the drug. After releasing the vacuum, the beads were weighed and collected for studying the elution kinetics in phosphate buffer solution (PBS). The excess antibiotic was discarded. The same weight of beads and same volume of antibiotics were replicated. The samples were divided into five groups. Group 1 HA beads were loaded with Gentamicin, Group 2 HA beads were loaded with Vancomycin, Group 3 HA beads were loaded with Amoxycillin clavulanate, Group 4 medium sized HASi beads were loaded with Amoxycillin clavulanate and Group 5 large sized HA Si beads were loaded with Amoxycillin clavulanate.

Antibiotic elution

Antibiotic impregnated beads were placed in 10 ml sterile glass tubes to which 5 ml of sterile phosphate buffer solution (PBS) was added. The tubes were incubated at 37°C. The time points chosen to determine elution was on a daily basis. Every 24 h the PBS was drained and collected for analysis. The tubes were replenished with fresh samples of PBS and the tubes were returned to the incubator. The element samples were stored at 4°C until analyzed together at the end of the final elution point of 15 days.

The quantity of antibiotics present in the elements was estimated by UV-visible spectrophotometer (UV/VIS spectrometer, Lambda 25 Perkin Elmer). Calibration curves were prepared by plotting drug concentration against the absorbance of standard solutions. Absorbance of the sample solutions was measured and from the calibration plot their concentrations were calculated. Antibiotic solutions prepared from the same stock as those used for loading the beads were used for preparing standard solutions. Depending on the concentrations, samples were analyzed either as such or after diluting so that the values fell within the linear range of standard curves. In the case of vancomycin and amoxicillin potassium clavulanate, absorbance of the clear solution was measured at λ max of 280 nm and 271 nm, respectively. For gentamicin the absorbance at λ max of 333 nm was measured after derivatising with o-phthaldehyde reagent.


   Results Top


[Figure 3] shows the mean amount of antibiotic released from 1 g of beads loaded with drugs at different elution point. All samples showed a general trend of exponential decay in eluted antibiotic amount with respect to time. The peak concentration was highest for amoxicillin-clavulanate and lowest for vancomycin. However, the release profile is not exactly identical for all the three drugs tested. A higher level of elution is maintained for a longer duration for gentamicin. Amoxicillin clavulanate showed a faster decay in the amount released. A general trend that is observed is that the drug release occurred in two stages: in the first stage (up to the end of the first week) especially in the initial phase of the release (up to the end of the first week) decayed rapidly and thereafter up to the end of the second week showed a steady release. In all cases, release of drug continued for more than 20 days.
Figure 3: Histogram with original elution values

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The data was transformed to loge [Figure 4], for making the distribution pattern symmetrical. The pattern showed that the distribution is not strictly symmetrical even after transformation. Hence, for comparison of the five groups together, a non-parametric ANOVA, that is, Kruskal–Wallis was applied [Table 1]. The results are presented accordingly with mean, median, standard deviation, interquartile range, and Box–whisker plots [Table 2] and [Figure 5]. The degrees of freedom of the test statistics (Chi square) is 4.
Figure 4: Histogram with log e values

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Table 1: Statistics showing mean, median, standard deviation, and range

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Table 2: ANOVA values

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Figure 5: Box Whisker Plot

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   Discussion Top


Enteral and parenteral drug deliveries are the traditional routes of administering therapeutic agents. However, they seldom provide targeted bioavailability and are beset with systemic side-effects. Tissue engineering attempts to circumvent these issues, by devising novel technologies to deliver therapeutic agents on site. Tissue engineering has been defined as an interdisciplinary field that applies the principles of engineering and life sciences, toward the development of biological substitutes that restore, maintain, or improve tissue function.[5]

In 1982, the FDA approved the investigation of porous HAp, produced through the hydrothermal transformation of Goniospora, a South Pacific Coral.[6] Biological HAp is a nanocrystalline, multi-substituted, carbonated hydroxyapatite, deficient in calcium and with a reduced number of structural hydroxyl groups. Synthetic HAp is osteoconductive, biodegradable, nontoxic, has high loading capacity and can be subject to ionic modifications that can endow it with antibacterial and osteointegrative properties. In 1987, the first bone targeted drug, WP-1 was designed, which combined tetracycline known for its affinity to bone and acetazolamide, a carbonic anhydrase inhibitor.[7] A system that releases a therapeutic agent at a preselected biological site in a controlled manner is called a targeted drug delivery system. Bone targeting moieties and drug carriers are integral components of targeted drug delivery systems.[8] HAp has been used as carriers for antibiotics, hormones, anti-inflammatory agents, proteins, siRNAs, miRNAs, SERMs, and anticancer drugs. These pharmacologic agents are attached to the HAp by chemical, physical, or mechanical linking; or adsorption.[9]

Targeted drug delivery systems enable a sustained local site concentration of the drug, without causing toxicity or drug levels falling below the minimum therapeutic range.[4] In bone that is affected by osteomyelitis, osteonecrosis, or osteoporosis, such delivery systems ensure that the drug reaches the target site, in spite of impaired vascularization. The added advantage of HAp based systems is its osteoconductive property which can restore and regenerate lost osseous tissue. HAp can form an additional layer of calcium phosphate on its surface, thus enabling tissue integration and eliminating fibrous tissue formation.[10]

PMMA incorporated with gentamicin, is a commercially available bone cement, popular among clinicians for treating bone infections like osteomyelitis. The antibiotic is released into the surgical site, but its nonbiodegradable nature mandates a second surgical intervention.[4] Its depolymerization is exothermic, which limits the variety of drugs that can be conjugated with it. Incomplete elution of the drug from the matrix is another liming factor.[9]

Gentamicin and vancomycin have been extensively studied for their drug elution profiles. Rauschmann et al. studied the release properties of gentamicin and vancomycin for a bioresorbable composite of calcium sulphate and nanoparticulate hydroxyapatite, as compared to pure calcium sulfate alone; where the former showed 94.7% vs. 95.8% elution for gentamicin and 96. 3% vs. 74.8% for vancomycin; over a period of 10 days.[11] HAp impregnated with vancomycin was used to treat rabbits with osteomyelitis and showed no histological evidence of infection on the 42nd day, thus proving their efficacy against MRSA or Staphylococus aureus, SCVs (small colony variants).[12] HAp microspheres with high porosities showed increased drug loading capacity ≈ 50% and increased initial release ≈ 65%, by improved ice-templated spray drying techniques (ITSD).[13]

A novel elastic hydroxyapatite glucan composite loaded with amikacin and gentamicin was tested against two gram-positive and two gram-negative bacterial stains. The loaded drugs acted in a biphasic mode, and was released within 48–119 h in a pore-dependent manner and inhibited bacterial growth in the culture medium.[14] Post-porous HAp coated Ti4Al4V prepared for electrolytic deposition of vancomycin-chitosan composite showed an initial burst of 55%, followed by steady release of 20% till day 5, and slower sustained release of 25% after day 6, resulting in a bacterial inhibition zone diameter of 30 mm, that lasted up to 30 days.[15]

High pressure consolidated calcium phosphate-polycaprolactone composite beads demonstratedin vitro release of vancomycin over a period of 4–11 weeks, without altering the structure and activity of the drug.[16] Vancomycin-loaded nanohydroxyapatite pellets, successfully repaired bone defects and controlled infection in MRSA-induced chronic osteomyelitis by maintaining the drug concentration for 12 weeks in bone and soft tissues.[17] Nanoporous, biomimetic hydroxyapatite coatings deposited on TiO2 coated fixation pins, adsorbed with tobramycin showed antibacterial activity against staphylococcus aureus for 8 days.[18]

HAp-based porous scaffold loaded with ceftriaxone sulbactam were found to release the drugin vitro studies andin vivo rabbit tibia osteomyelitis model even after 42 days, against staphylococcus aureus.[19]

Zigecycline and HAp were compressed into tablets and drug release was evaluated in PBS solution by UV-vis spectrophotometry. Elution was detected up to 30 days.[20] Zigecycline impregnated HAp was implanted in adult Wistar albino rat tibias with MRSA. Histopathologic disease severity scores and bacterial counts demonstrated that the drug was eluted effectively from the carrier.

This study was intended to demonstrate the use of HA and HASi nanoporous beads as post-loaded drug eluting platforms and the effect of different type of antibiotic molecules on the release kinetics of the loaded drug. The release profile of all the three drugs is characterized by a biphasic behavior. Group 1 beads eluted 54,526.47 μg/g on day 1 and continued till day 24 (65.42 μg/g). Group 2 released 25110 μg/g on day 1 and continued till day 21 (16 μg/g). Group 3 eluted 38,923.89 μg/g on day 1 and continued till day 17 (6.01 μg/g). Group 4 released 27,771.10 μg/g on day 1 and continued till day 17 (4.79 μg/g). Group 5 eluted 38,534.27 μg/g on day 1 and continued till day 17 (5.83 μg/g). Drug elution values exceeded the bactericidal concentration levels for all drugs tested.

Bioceramic drug carrying platforms have their role in the comprehensive treatment applications of maxillofacial osseous lesions like cysts and tumors in the postsurgical phase. Their capacity can be harnessed to carry tailor-made pharmaceutical agents into the bony defects, effect adequate elution, and allow for osteoconduction, thereby promoting osseous healing.

Financial support and sponsorship

Nil.

Conflict of interest

There is no conflict of interest.



 
   References Top

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Rauschmann M, Wichelhaus TA, Stirnal V, Dingeldein E, Zichner L, Schnettler R, Alt V. Nanocrystalline hydroxapatite and calcium sulphate as biodegradable composite carrier material for local delivery of antibiotics in bone infections. Biomaterials 2005;26:2677-84.  Back to cited text no. 11
    
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Yu M, Zhou K, Li Z, Zhang D. Preparation, characterization and in vitro gentamicin release of porous HA microspheres, Mater. Sci Eng C 2014;45:306-2.  Back to cited text no. 13
    
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A, Zima A, Binalska G. Biphasic mode of antibacterial action of aminoglycoside antibiotics-loaded elastic hydroxyapatite –glucan composite. Int J Pharm 2013;454:285-95.  Back to cited text no. 14
    
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Yang CC, Lin CC, Liao JW, Yen SK. Vancomycin-chitosan implant for drug controlled release. Mater Sci Eng C 2013;33: 2203-12.  Back to cited text no. 15
    
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Makarov C, Cohen V, Raz-Pasteur A, Gotman I. In vitro elution of vancomycin from biodegradable osteoconductive calcium phosphate-polycaprolactone composite beads for treatment of osteomyelitis, Eur I Pharm Sci 2014;62:49-56.  Back to cited text no. 16
    
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Jiang JL, Li YF, Fang TL, Zhou J, Li XL, Wang YC, Dong J. Vancomycin –loaded porous hydroxyapatite pellets to treat MRSA-induced chronic osteomyelitis with bone defect in rabbits. Inflamn Res 2012;61:207-15.  Back to cited text no. 17
    
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Lilija M, Sorensen JH, Brohede U, Astrand M, Procter P, Arnoldi J, et al. Drug loading and release of tobramycin from hydroxyapatite coated fixation pins. J Mater Sci Mater Med 2013;24:265-74.  Back to cited text no. 18
    
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Kundu B, Soundrapandian C, Nandi SK, Mukherjee P, Dandapat N, Roy S, et al. Development of new localized drug delivery system based on ceftriaxone-sulbactam composite drug impregnated porous hydroxyapatite: A systematic approach for in vitro and in vivo animal trial. Pharm Res 2010;27:1659-76.  Back to cited text no. 19
    
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Colovic B, Pasalic S, Jokanovic V. Influence of hydroxyapatite pore geometry on tigecycline release kinetics. Ceram Int 2012; 38:6181-9.  Back to cited text no. 20
    

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Correspondence Address:
Dr. Deepti Simon
Department of Oral and Maxillofacial Surgery, Government Dental College, Thiruvananthapuram, Kerala
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/ijdr.IJDR_357_19

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