Angelic Guarda MSC Thesis

RELEASE AND BIOACTIVITY OF ANTIBIOTICS FROM INJECTABLE
POLYMERIC DCPD CEMENT

by
ANGÉLICA GUARDIA
THESIS
Submitted to the Graduate School
of Wayne State University,
Detroit, Michigan
in partial fulfillment of the requirements
for the degree of
MASTER OF SCIENCE
2020
MAJOR: BIOMEDICAL ENGINEERING
Approved By:
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Advisor Date
ProQuest Number: 27957544
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ACKNOWLEDGEMENTS
I would like to thank my family and close friends for their continued support. I would also
like to thank Dr. Weiping Ren for providing this research opportunity and guidance throughout
the project.
ii
TABLE OF CONTENTS
ACKNOWLEDGEMENTS………………………………………………………………………ii
LIST OF TABLES .................................................................................................................................... vi
LIST OF FIGURES ................................................................................................................................. vii
CHAPTER 1 – GENERAL INTRODUCTION ..................................................................................... 1
1.1 Brief Overview of Bone Anatomy and Physiology .............................................................. 1
1.2 Pathophysiology of Osteomyelitis ........................................................................................ 1
1.3 Antibiotic-loaded Bone Cements as Osteomyelitis Treatment ............................................. 3
CHAPTER 2 – PHYSICOCHEMICAL CHARACTERIZATION OF ANTIBIOTIC-LOADED

P-DCPD CEMENT ....................................................................................................................... 5
2.1 Introduction ........................................................................................................................... 5
2.1.1 Brief Overview of Calcium Phosphate Cements as Drug Delivery Vehicles ................ 5
2.1.2 Advantages of P-DCPD Cement .................................................................................... 7
2.2 Materials and Methods .......................................................................................................... 9
2.2.1 Preparation of CPP powder and ACPP hydrogel ........................................................... 9
2.2.2. Preparation of TTCP powder ......................................................................................... 9
2.2.3 Preparation of Drug-loaded P-DCPD Cement Samples ............................................... 10
2.2.3 Setting Time and Injectability ...................................................................................... 10
2.2.4 Zeta Potential ................................................................................................................ 10
2.2.5 Mechanical Testing (Compressive Modulus and Compressive Strength) .................... 10
2.2.6 Cement Degradation ..................................................................................................... 10
2.2.7 Cumulative Drug Release and Drug Concentration ..................................................... 11
iii
2.2.8 Statistical Analysis ....................................................................................................... 12
2.3 Results ................................................................................................................................. 12
2.3.1 Setting Time and Injectability ...................................................................................... 12
2.3.2 Zeta Potential ................................................................................................................ 13
2.3.3 Mechanical Testing (Compressive Modulus and Compressive Strength) .................... 14
2.3.5 Cement Degradation ..................................................................................................... 16
2.3.4 Cumulative Drug Release and Drug Concentration ..................................................... 18
2.4 Discussion ........................................................................................................................... 22
CHAPTER 3 – ANTIBACTERIAL EFFICACY OF ANTIBIOTIC-LOADED P-DCPD

CEMENT...................................................................................................................................... 27
3.1 Introduction ......................................................................................................................... 27
3.2 Materials and Methods ........................................................................................................ 27
3.2.1 Bacterial Culture ........................................................................................................... 27
3.2.2 Modified antibiotic minimum inhibitory concentration (MIC) assay .......................... 27
3.2.3 Statistical Analysis ....................................................................................................... 28
3.3 Results ................................................................................................................................. 28
3.3.1 Drug-loaded P-DCPD Cement Discs ........................................................................... 28
3.3.2 Drug Eluent................................................................................................................... 30
3.3.3 Degraded P-DCPD Powder Discs ................................................................................ 32
3.4 Discussion ........................................................................................................................... 33
CHAPTER 4 – CYTOCOMPATIBILITY OF ANTIBIOTIC-LOADED P-DCDP CEMENTS.. 35
4.1 Introduction ......................................................................................................................... 35
iv
4.2 Materials and Methods ........................................................................................................ 35
4.2.1 Cell Seeding and Culture .............................................................................................. 35
4.2.2 Cell Proliferation Assay (MTT) ................................................................................... 35
4.2.3 Cell Toxicity Assay (LDH) .......................................................................................... 36
4.3 Results ................................................................................................................................. 36
4.3.1 Cell Proliferation (MTT) .............................................................................................. 36
4.3.2 Cytotoxicity (LDH) ...................................................................................................... 39
4.4 Discussion ........................................................................................................................... 42
CHAPTER 5 – CONCLUSION AND FUTURE WORK .................................................................. 44
APPENDIX: ABBREVIATIONS .......................................................................................................... 46
REFERENCES ......................................................................................................................................... 47
ABSTRACT .............................................................................................................................................. 51
AUTOBIOGRAPHICAL STATEMENT ............................................................................................. 53
v
LIST OF TABLES
Table 1. Setting time and injectability of EM-doped P-DCPD cement. ....................................... 12
Table 2. Setting time and injectability of antibiotic-loaded P-DCPD cements ............................ 12
Table 3. Zeta potential of EM-doped P-DCPD cement particles.................................................. 13
Table 4. Zeta potential of antibiotic-loaded P-DCPD cement particles........................................ 13
Table 5. Compressive strength and compressive modulus of EM-doped P-DCDP cement ......... 14
Table 6. Compressive strength and compressive modulus of antibiotic-loaded P-DCPD scaffolds.

........................................................................................................................................... 15
vi
LIST OF FIGURES
Figure 1. Stress-strain curves for antibiotic-loaded P-DCPD cement scaffolds. .......................... 14
Figure 2. Compressive modulus of antibiotic-loaded P-DCDP cement scaffolds. Asterisk (*)

denotes statistically significant difference (p < 0.05) to drug-free cement. Error bars
represent SD. ..................................................................................................................... 15
Figure 3. Compressive strength of antibiotic-loaded P-DCDP cement scaffolds. Asterisk (*)

denotes statistically significant difference (p < 0.05) to drug-free cement. Error bars
represent SD. ..................................................................................................................... 16
Figure 4. Degradation of EM-loaded P-DCDP cement scaffolds. Asterisk (*) denotes statistically
significant difference (p < 0.05) to drug-free cement. Error bars represent SD. .............. 16
Figure 5. Degradation of antibiotic-loaded P-DCDP cement scaffolds. Asterisk (*) denotes

statistically significant difference (p < 0.05) to drug-free cement. Error bars represent SD.
........................................................................................................................................... 17
Figure 6. In vitro degradation of antibiotic-loaded P-DCDP cement scaffolds. ........................... 17
Figure 7. Cumulative drug release of EM-doped P-DCPD cement. Asterisk (*) denotes statistically
significant difference (p < 0.05) between EM and all other groups. Error bars represent SD.
........................................................................................................................................... 18
Figure 8. Cumulative drug release of EM-doped P-DCPD cement for first 72 hours. Asterisk (*)
denotes statistically significant difference (p < 0.05) between EM and all other groups.
Error bars represent SD. .................................................................................................... 18
Figure 9. Eluent drug concentration of EM-doped P-DCPD cement. Asterisk (*) denotes
statistically significant difference (p < 0.05) between EM and all other groups. Error bars
represent SD. ..................................................................................................................... 19
Figure 10. Eluent drug concentration of EM-doped P-DCPD cement for the first 72 hours. Asterisk
(*) denotes statistically significant difference (p < 0.05) between EM and all other groups.
Error bars represent SD. .................................................................................................... 19
Figure 11. Cumulative drug release of antibiotic-doped P-DCPD cement. Asterisk (*) denotes
statistically significant difference (p < 0.05) between groups. Error bars represent SD. . 20
Figure 12. Eluent drug concentration of Van and Tob-doped P-DCPD cement. Asterisk (*) denotes
statistically significant difference (p < 0.05) between groups. Error bars represent SD. . 20
vii
Figure 13. Eluent drug concentration of Van and Tob-doped P-DCPD cement for the first 72 hours.
Asterisk (*) denotes statistically significant difference (p < 0.05) between groups Error
bars represent SD. ............................................................................................................. 21
Figure 14. Individual eluent drug concentration of Van/Tob-doped P-DCPD cement. Asterisk (*)
denotes statistically significant difference (p < 0.05) between groups. Error bars represent
SD. .................................................................................................................................... 21
Figure 15. µ-CT scans of antibiotic-loaded P-DCPD cement discs. ............................................ 24
Figure 16. Antibiotic-loaded P-DCPD cement disc time course. ................................................. 29
Figure 17. EM-doped P-DCPD cement ZOI. Asterisk (*) denotes statistically significant difference
(p < 0.05) between EM and all other groups. Error bars represent SD. Error bars represent
SD. .................................................................................................................................... 29
Figure 18. Antibiotic-doped P-DCPD cements ZOI. Asterisk (*) denotes statistically significant
difference (p < 0.05) to Tob. Error bars represent SD. ..................................................... 30
Figure 19. Drug eluent-soaked paper discs for bacterial inhibition assay. ................................... 30
Figure 20. EM-doped P-DCPD eluent ZOI. ................................................................................. 31
Figure 21. Antibiotic-doped P-DCPD eluent ZOI. Error bars represent SD. ............................... 31
Figure 22. Degraded P-DCPD powder discs for bacterial inhibition assay. ................................. 32
Figure 23. Antibiotic-doped degraded P-DCPD powder discs ZOI. Asterisk (*) denotes statistically
significant difference (p < 0.05) to all other groups. Error bars represent SD. ................ 32
Figure 24. Impact of EM-doped P-DCPD eluent on cell proliferation. Asterisk (*) denotes
statistically significant difference (p < 0.05) to drug-free P-DCPD eluent. Error bars
represent SD. ..................................................................................................................... 36
Figure 25. Impact of EM-doped P-DCPD particles on cell proliferation. Asterisk (*) denotes
statistically significant difference (p < 0.05) to drug-free P-DCPD particles. Error bars
represent SD. ..................................................................................................................... 37
Figure 26. Impact of degraded EM-doped P-DCPD particles on cell proliferation. Line over bars
denotes no statistically significant difference between groups. Error bars represent SD. 37
Figure 27. Impact of antibiotic-doped P-DCPD eluent on cell proliferation. Line over bars denotes
no statistically significant difference between groups. Error bars represent SD. ............. 38
viii
Figure 28. Impact of antibiotic-doped P-DCPD particles on cell proliferation. Line over bars
Figure 29. Impact of degraded antibiotic-doped P-DCPD particles on cell proliferation. Line over
bars denotes no statistically significant difference between groups. Error bars represent SD.
........................................................................................................................................... 38
Figure 30. Impact of EM-doped P-DCPD eluent on cell toxicity. Asterisk (*) denotes statistically
significant difference (p < 0.05) to drug-free P-DCPD eluent. Error bars represent SD. . 39
Figure 31. Impact of EM-doped P-DCPD particles on cell toxicity. Asterisk (*) denotes statistically
significant difference (p < 0.05) to drug-free P-DCPD particles. Error bars represent SD.
........................................................................................................................................... 40
Figure 32. Impact of degraded EM-doped P-DCPD particles on cell toxicity. Line over bars
Figure 33. Impact of antibiotic-doped P-DCPD eluent on cell toxicity. Asterisk (*) denotes
statistically significant difference (p < 0.05) to drug-free P-DCPD eluent. Error bars
represent SD. ..................................................................................................................... 40
Figure 34. Impact of antibiotic-doped P-DCPD particles on cell toxicity. Line over bars denotes
no statistically significant difference between groups. Error bars represent SD. ............. 41
Figure 35. Impact of degraded antibiotic-doped P-DCPD particles on cell proliferation. Asterisk
(*) denotes statistically significant difference (p < 0.05) to degraded drug-free P-DCPD
particles. Error bars represent SD. .................................................................................... 41
ix
1
CHAPTER 1 – GENERAL INTRODUCTION
1.1 Brief Overview of Bone Anatomy and Physiology
Bone is hard, largely calcareous connective tissue that composes most of the human
skeleton [1]. In addition to performing protective and locomotive functions, bone plays an
important role in maintaining acid-base balance and mineral homeostasis, acts as a depot for
cytokines and growth factors, and hosts hematopoiesis within its marrow [2].
In order to adapt to changes in biomechanical stress, bone undergoes constant remodeling
during its lifetime. In this process, old, damaged bone is replaced with new bone which is
mechanically stronger [2]. This remodeling process is carried out by osteoblasts, which deposit
new bone, and osteoclasts, which break down old bone [2].
Bone can be classified into four general categories: long bones, short bones, flat bones and
irregular bones [2]. Examples of long bones include the humeri, femurs, and tibiae [2]. Long bones
are composed of a hollow shaft known as diaphysis, primarily made up of dense cortical bone that
surrounds the marrow space, and rounded ends known as epiphyses, which are composed of porous
trabecular bone surrounded by a relatively thin layer of cortical bone [2]. The inner surface of the
cortical bone is surrounded by the endosteum, a vascularized membranous structure, while the
outer cortical surface of the bone is surrounded by the periosteum, a fibrous connective tissue
sheath that contains nerve fibers, blood vessels, osteoclasts and osteoblasts [2].
1.2 Pathophysiology of Osteomyelitis
Osteomyelitis is defined as an inflammatory process in the bone and bone marrow caused
by bacteria that often results in local bone loss and necrosis; in short, the term implies infection of
the bone or joint where the infection can be limited to a portion of the bone or affect several regions
[3, 4]. Patients receiving joint replacements, also known as arthroplasties, are one of the groups
2
most vulnerable to infection: the incidence of osteomyelitis following arthroplasty ranges from
0.3% to 2.4% for total hip arthroplasties and 1% to 3% for total knee arthroplasties [3]. A
Nationwide Inpatient Sample (NIS) study concluded that joint infection made up 25% of revision
arthroplasties in the knee and 15.4% of revision arthroplasties in the hip in the United States, where
the average individual hospitalization costs related to periprosthetic infection ranged from $25,692
to $31,753 [3]. In general, the overall incidence of osteomyelitis and consequent revision
arthroplasties may increase due to patient risk factors like diabetes, age, peripheral vascular
disease, intravenous drug use and immunodeficiency due to disease or treatment [3, 4].
Osteomyelitis can spread according to three general mechanisms: hematogenous spread,
infection associated with vascular or neurologic insufficiency and contiguous contamination [3].
Contiguous infection, the focus of this brief overview, is an infection spread from a contaminated
site, and the main mechanism in which osteomyelitis occurs in patients undergoing surgery where
there is direct contamination of bacteria in open fractures or via contaminated implants during
arthroplasty [3]. It is estimated that 80% to 90% of cases are caused by Staphylococcus aureus (S.
aureus) [3].
Once the bone is infected and the bacteria induce an acute inflammatory reaction, the
infection affects the periosteum and spreads within the bone resulting in necrosis [3]. In order to
fight the pathogen, leukocytes release cytokines and enzymes that break down infected tissue;
consequently, purulence consisting of dead leukocytes, host cells and bacteria fills intercellular
spaces around the infection and causes an abscess [4]. Lifting of the periosteum due to abscess
growth impairs blood supply to the affected area and results in segmental bone death known as a
sequestrum, and this vascular impairment isolates the focus of infection from the immune system
and systemic antibiotic treatments [3, 4]. Once in the chronic stage of infection, inflammatory cells
3
and their cytokine release stimulate bone resorption by osteoclasts, ingrowth of fibrous tissue and
the deposition of new bone in the surrounding area, where this newly deposited bone may form a
layer of living tissue around the necrotic, infected bone, known as involucrum [3]. Continuous
growth of the abscess may lead to excess purulence eventually escaping through an opening in the
bone known as sinus tract [3, 4].
1.3 Antibiotic-loaded Bone Cements as Osteomyelitis Treatment
It has been established that the success of osteomyelitis treatment is heavily reliant on the
debridement of infected tissue and removal of any necrotic bone prior to beginning an
antimicrobial treatment, with the purpose of leaving only healthy, viable tissue to accelerate the
restoration of function [4, 5]. Still, because it cannot be assumed that all necrotic tissue will be
successfully removed, debridement must be combined with antibiotic therapy [5]. However,
antibiotics are poor penetrators of necrotic bone, meaning that systemic administration of a high
enough dose to penetrate and clear the infection in the necrotic region will often result in toxicity
to the patient, and the antibiotic course typically lasts between 4 to 8 weeks [4, 5]. Consequently,
a material that allows the local delivery of antibiotics to the site of infection while also promoting
the regeneration of bone would be ideal for the treatment of this disease.
Poly-methylmethacrylate (PMMA) cement is the standard vehicle for localized antibiotic
delivery in osteomyelitis treatment; as an injectable space filler or in the form of beads, PMMA
cement can be combined with heat-stable antibiotics like vancomycin, tobramycin and gentamycin
[3, 6]. However, PMMA has several limitations as a drug delivery vehicle. Due to its high
exothermic property, PMMA can only be mixed with heat stable antibiotics, limiting its
application, and its drug elution is poor [3]. There are concerns regarding the toxicity of leached
MMA monomer from the cement, which may cause systemic effects like hypoxia as well as local
4
tissue toxicity [3]. Lastly, PMMA cement is non-biodegradable, meaning that it requires a second
surgical intervention for removal, increasing cost to the patient and the possibility of reinfection.
[3, 4]
These obstacles can be overcome by using biodegradable drug delivery vehicles like
calcium sulfate or, in particular, calcium phosphate cements [4]. These tailorable, biodegradable
materials boast a chemical composition similar to that of native bone tissue, are self-hardening and
can be injectable or molded as beads or scaffolds that can fit the shape of the defect [7]. Calcium
phosphate cements not only allow for the local delivery of a wider variety of antibiotics by
avoiding the issue of exothermic reaction, but also provide a promising scaffold for bone repair
and regeneration [4]. Thus, in the following chapter and throughout the rest of this thesis, the focus
will remain on calcium phosphate cements: their physicochemical properties, their applications
and limitations.
5
CHAPTER 2 – PHYSICOCHEMICAL CHARACTERIZATION OF ANTIBIOTIC-

LOADED P-DCPD CEMENT
2.1 Introduction
2.1.1 Brief Overview of Calcium Phosphate Cements as Drug Delivery Vehicles
Calcium phosphate is present in bone tissue and teeth and has become a material of interest
in the repair of skeletal defects [7]. Calcium phosphate cements (CPC) are made up of one or more
solid calcium phosphate compounds combined with a liquid phase [7]. By mixing the powders and
liquid in a suitable ratio, one will obtain a self-setting paste that hardens at room temperature due
to the entrapment of crystals precipitated during the mixture reaction [7]. Commonly used calcium
phosphate compounds in the preparation of CPC include monocalcium phosphate monohydrate
(MCPM), dicalcium phosphate anhydrous (DCPA), dicalcium phosphate dihydrate (DCPD),
tetracalcium phosphate (TTCP), and amorphous calcium phosphate (ACP), among others. [7]
CPC can be divided into two categories according to its hydration products, which
ultimately depends on the pH of the cement paste: hydroxyapatite (HA) result at a pH above 7,
while DCPD result at a pH below 6 [7]. The resulting type of cement is what ultimately determines
its solubility both in vitro and in vivo [7]. While sintered HA bears great crystallographic and
compositional similarities to bone mineral, this type of cement is essentially inert non-
biodegradable [8]. DCPD cement, on the other hand, is fully resorbable in vivo and can stimulate
cellular repair at the molecular level, making it an appropriate material for the creation of bone
regeneration scaffolds and localized drug delivery in the treatment of musculoskeletal disorders
like osteomyelitis [7]. DCPD cements are made up of the combination of an acidic calcium
phosphate component, like MCPM, with an alkaline calcium phosphate component like TTCP
along with a liquid phase, in many cases water [7]. Due to its excellent biodegradable properties,
DCPD has been the CPC of choice for this project.

6
In the context of the medical applications of DCPD cements, injectability is a crucial factor
because it allows the surgeon to inject the paste such that it can adapt to the shape of any bone
defect and then set in place [7]. The injectability of DCPD cements can be modified or improved
by adjusting the liquid-to-powder ratio and adding citrate ions or viscous polymer solutions;
particle shape also influences the result, with round particles generally improving injectability [7].
The cement setting and working time can also be adjusted via changes in its powder composition,
powder-to-liquid ratio and particle shape and size [7]. For any cementitious application, but
particularly in those in which the cement must be injectable, the setting and working time are
crucial factors: a long setting time prevents the cement from supporting stresses or solidifying its
shape within a feasible time interval, and a short working time eliminates the possibility of
adjustments in placement [7]. Thus, while there is no established standard for DCPD cement
setting and working times, the literature suggests 10-15 min as a suitable setting time and 6-10
min as a suitable working time [7].
If it is to be used for a clinical application, the cement in question should have a strength
and modulus comparable to the bone being replaced to prevent stress shielding and fractures or
complications [7]. However, DCPD cements are typically limited to low or non-load-bearing
applications due to the inherent brittleness and low strength of these type of cements [7]; thus,
there is great interest in improving the mechanical properties of DCPD cements.
Ideally, bone cements should also provide support for the deposition of bone matrix by
osteoprogenitor cells. With CPC in general, this process is affected not only by the scaffold’s
architecture but also by the rate at which the scaffold is resorbed, which should be slow enough to
allow new bone tissue to grow within the scaffold [7]. This resorption rate is directly related to the
scaffold’s crystallinity and porosity; cements with lower degree of crystallinity like DCPD are able
7
to resorb faster than HA cements [7]. While most CPC formulations have a porosity between 30%-
50% and permit the transport of nutrients and waste, the size of these pores is typically not large
enough to allow tissue ingrowth [7]. Thus, while osteoinductivity is desired, it is generally
accepted that DCPD cement can act as an osteoconductive material but not an osteoinductive one
[7].
The potential of DCPD cements as drug delivery vehicles is of great interest. Unlike
PMMA cements, DCPD cements can set at body temperature and are not as limited in the variety
of drugs, proteins or drug factors that can be incorporated into their matrix, making them attractive
candidates for the treatment of osteomyelitis [7]. Due to its micro/nanoporous structure, DCPD
cements can avoid burst drug release, though the release rate and amount of drug will ultimately
depend on the overall cement formulation as well as the physicochemical properties of the drug
itself [7].
Although DCPD cements can be produced in large quantities with relatively low cost and
have the potential to become an off-the-shelf product, there are several drawbacks associated with
this material. The poor mechanical properties of DCPD, specifically its high brittleness and
comparatively low strength, is one of its most notable limitations as well as the material’s lack of
osteoinductivity and finicky handling properties, which also present challenges in the clinical
setting [8].
2.1.2 Advantages of P-DCPD Cement
We defined a poly-dicalcium phosphate dihydrate (P-DCPD) cement as a formulation in
which amorphous calcium polyphosphate (ACPP) gel is used as the acidic calcium source in the
preparation of DCPD. The structure of calcium polyphosphate (CPP) differs from other calcium
phosphate minerals like HA or tricalcium phosphate in that CPP is composed of linear

8
polyphosphate chains [9]. The structure of CPP can be either amorphous or crystalline depending
on the processing settings [9]. CPP is eventually hydrolyzed into the naturally occurring calcium
orthophosphate [9]. The key molecular structural difference between P-DCPD and classic DCPD
is that P-DCPD is composed of interconnected P-DCPD crystal particles interlocked to the
polyphosphate chains, while the latter is composed of a packed DCPD crystal particles held by
weak mutual bonding [9]. The advantages of this new formulation are mainly due to the unique
microstructure being formed based on the new self-setting system of CPP gel matrix, and include
improved mechanical strength, good injectability and superior cohesion [9]. The combination of
moldability, self-setting nature, lack of noxious byproducts, biocompatibility, and potential for
osteoconduction make P-DCPD a promising material for dental and non-load-bearing orthopedic
applications.
The ability of linear CPP gel matrix to delay antibiotic release has been demonstrated by
Dion et al, where vancomycin release from CPP gel matrices was significantly reduced during the
first 2 to 4 h of elution, while retaining its bactericidal activity [9, 10]. Chehreganianzabi et al also
showed that loading antibiotic in fully formed CPP hydrogel enhances the antibiotic retention
within the matrix, resulting in reduced burst release of embedded drugs [11]. Therefore, in this
project, we propose a method to prepare injectable and self-setting antibiotic-loaded P-DCPD
cement via the reaction of CPP gel and TTCP to overcome the main limitations of CPC in
therapeutic applications, mainly, weak mechanical properties and drug burst release.
The purpose of this chapter is to investigate the effect of antibiotic loading on the
physicochemical characteristics and drug release behavior of P-DCPD. The cement samples were
loaded with erythromycin (EM), vancomycin (Van), tobramycin (Tob), and equal parts of
vancomycin and tobramycin (Van/Tob), all at same concentration (10% w/w). EM is a

9
hydrophobic antibiotic with reported ability to enhance bone formation [12, 13]. Van and Tob are
hydrophilic antibiotics widely used in the treatment of osteomyelitis [14, 15, 16]. Overall, these
antibiotics were selected due to their widespread use and effectiveness against infections caused
by methicillin-resistant forms of Staphylococcus aureus (MRSA).
2.2 Materials and Methods
2.2.1 Preparation of CPP powder and ACPP hydrogel
Calcium phosphate monobasic monohydrate (Ca[H2PO4]2:H2O; Sigma-Aldrich, St. Louis,
MO) was calcined for 10 h at 500 ℃ followed by melting for 1 h at 1200 ℃ to produce an
amorphous glass and quenched at room temperature for cooling. The material was hammered to
make small particles prior to grind in a planetary ball mill (Across International, Berkeley, NJ).
The resulting particles were sieved (Laboratory Test Sieves, Fisher Scientific, Pittsburg, PA)
continuously to segregate particles less than 75 μm. Prior to usage, CPP powders were stored in a
vacuum desiccator to avoid moisture absorption.
For hydrogel preparation, CPP powder was added to distilled water (0.1 g ml−1), stirred for
90 min at 700 rpm at room temperature (25 ℃) and suspended for 24 h until the produced gel
precipitated and the water phase become clear.
2.2.2. Preparation of TTCP powder
TTCP powder was prepared by solid-state reaction of DCPA (CaHPO4) and calcium
carbonate (CaCO3) (Sigma- Aldrich) by heating at 1500 °C for 18 h as stated in [17]. The resulting
particles were sieved (Laboratory Test Sieves, Fisher Scientific, Pittsburg, PA) continuously to
segregate particles less than 75 μm. Prior to usage, TTCP powders were stored in a vacuum
desiccator to avoid moisture absorption.

10
2.2.3 Preparation of Drug-loaded P-DCPD Cement Samples
P-DCPD cement was prepared by mixing CPP hydrogel with TTCP and sodium citrate
(Na3C6H5O7) (ADM Evolution Chemicals, Chicago, IL) (weight ratio of 1:0.87:0.15, w/w) along
with 240 μl gel supernatant at room temperature. P-DCPD cements were loaded with 10%
erythromycin (Sigma-Aldrich, St. Louis, MO, EM-P), 10% of vancomycin hydrochloride (NDC
Technologies, Dayton, OH, Van-P), 10% of tobramycin sulfate (X Gen Pharmaceuticals,
Horsehead, NY, Tob-P), and both 5% of Van and 5% Tob in combination (Van/Tob-P),
respectively, by mixing drug powders with CPP gel prior to adding TCPP for setting. P-DCPD
without drug loading was included as control.
2.2.3 Setting Time and Injectability
The setting time was measured using standard Gillmore needle method (Humboldt Mfg.
Co., Elgin, IL). The value of initial setting time was recorded.
2.2.4 Zeta Potential
The zeta potential of P-DCPD cement particles was measured with a Zetasizer (Nano-ZS;
Malvern Instruments, Malvern, UK) with built in Dispersion Technology Software.
2.2.5 Mechanical Testing (Compressive Modulus and Compressive Strength)
Cylindrical P-DCPD scaffolds (6 × 12 mm) were tested in axial compression using a
universal servo-hydraulic test machine (Instron 4302, MA) at a speed of 0.1 mm/s.
2.2.6 Cement Degradation
P-DCPD cement was molded into discs (8 × 3 mm). The discs were submerged in PBS
(Sigma-Aldrich) at room temperature for 28 days. Sample masses were measured prior to and post
11
immersion in PBS. Samples were allowed to dry in an oven at 60 °C for approximately 24 h before
recording their mass following immersion. Weight loss (%) was presented as:
Δ W = (W1 – W2)/W1 × 100
W1—weight before immersion, W2—weight after immersion
2.2.7 Cumulative Drug Release and Drug Concentration
P-DCPD cement was molded into discs (8 × 3 mm). The discs were submerged into PBS
at room temperature for 28 days. The supernatant was collected and replaced with fresh PBS at 2
h, 6 h, 1, 3, 7, 14, 21 and 28 days. The collected supernatant aliquots were stored at 4 ℃ and also
used for cell culture experiments and antimicrobial activity assays.
EM concentration was determined at 485 nm using regular 96-well plates by adding 100
μl of 3 M sulfuric acid and 100 μl of supernatant to the plate and by heating it up to 50 ℃ for 30
min as well as by warming the plate reader up to the same degree in advance.
Van concentration was determined in UV plate by loading 100 μl of the sample solution in
the UV plate with ultra-pure water as the assay solution and absorbance reading at 295 nm.
The concentration of Tob eluted was measured as described in [18]. Briefly, to prepare
sample solutions for the measurement of Tob concentration, 500 μl of each drug elution aliquot
was mixed with 500 μl acetylacetone-formaldehyde solution in a glass tube, placed in a boiling
water bath for 20 min and cooled to room temperature. The mixture was then diluted to 5 ml with
distilled water. The fluorescent intensity of 100 μl of the sample solution was measured at 460 nm
after excitation at 360 nm and converted to concentration using a standard curve of known Tob
concentrations.
12
2.2.8 Statistical Analysis
Microsoft Excel was used in the statistical analysis. Mean and standard deviation were
calculated from the experiment results. One-way ANOVA was used to analyze the difference
between group means. Statistical significance was set to be p < 0.05.
2.3 Results
2.3.1 Setting Time and Injectability
Table 1. Setting time and injectability of EM-doped P-DCPD cement.
P-DCPD EM-P
Setting time (min) (n = 1) 16 58
Injectability Intrusion (mm) (n = 4) 2.25±1.5 0.25±0.5*
As shown in Table 1, the addition of EM significantly extended EM-P setting time as
compared to P-DCPD. Nevertheless, EM-P cement is still injectable.
Table 2. Setting time and injectability of antibiotic-loaded P-DCPD cements
P-DCPD Van-P Tob-P Van/Tob-P
Setting time (min) (n=1) 16 14 77 12
Injectability (intrusion (mm) (n=4) 2.25±1.5 0.63±0.72 1.23±0.05 n/a
As shown in Table 2, Van-P and Van/Tob-P had a slightly reduced setting time as
compared to P-DCPD. However, Tob-P had a notably longer setting time. The injectability of P-
DCPD was slightly reduced compared to either Van-P or Tob-P (p > 0.05). However, the potential
for injectability still remains.

13
2.3.2 Zeta Potential
Table 3. Zeta potential of EM-doped P-DCPD cement particles.
P-DCPD EM-P
Zeta potential(mV) (n=5) -26.58 ± 1.48 -24.8 ± 0.57
As shown in Table 3, the zeta potential of EM-P slightly increased as compared to control.
However, the difference was not statistically significant.
Table 4. Zeta potential of antibiotic-loaded P-DCPD cement particles.
Zeta potential
-26.58 ± 1.48 -27.42 ± 0.68 -21.16 ± 1.01* -23.44 ± 0.97*
(mV) (n=5)
Data from contact angle measurement showed that P-DCPD is highly hydrophilic, and its
hydrophilicity was not influenced by loading of the drugs (data not shown). As shown in Table 4,
Zeta potential of P-DCPD was not influenced by the addition of Van. However, the zeta potential
was significantly reduced in both Tob-P (-21.16 ± 1.01) and Van/Tob-P (-23.44 ± 0.97), as
compared to P-DCPD (-26.58 ± 1.48), (both p < 0.05). In addition, there is a significant difference
of zeta potential between Tob-P and Van-P (p < 0.05).

14
2.3.3 Mechanical Testing (Compressive Modulus and Compressive Strength)
n=1
Chart Title
60
50
Stress (MPa)
40 Tob-P
P-DCPD
30
EM-P
20
Van/Tob-P
10 Van-P
0
0 0.005 0.01 0.015 0.02 0.025
Strain
Figure 1. Stress-strain curves for antibiotic-loaded P-DCPD cement scaffolds.
Table 5. Compressive strength and compressive modulus of EM-doped P-DCDP cement
P-DCPD EM-P
Compressive strength (MPa) (n = 3) 53.09±8.64 60.67±2.54
Compressive modulus (MPa) (n = 3) 3170.72±165.37 2927.20 ±11.56
Fig. 1 shows the stress-strain curve from the axial compression test of one scaffold
from each group. As shown in Table 5, EM-P showed a slight increase in the mechanical strength
and a slight reduction of the compressive modulus to some extent. However, the changes were not
statistically significant. Our data indicates that EM doping at current concentration did not result
in inferior mechanical strength and its impact on the compressive modulus was very slight (Fig. 2,
Fig. 3).
15
Table 6. Compressive strength and compressive modulus of antibiotic-loaded P-DCPD scaffolds.
Compressive strength
53.09±8.64 22.79±2.95 60.11±0.22 42.12±0.98
(MPa) (n =3)
Our data indicates that Tob doping slightly increased the mechanical strength and the
compressive modulus, while Van doping significantly reduced the mechanical strength as well as
the compressive modulus (Table 6, Fig. 2, Fig. 3). It seems that the impact of reduction of
mechanical strength is in part minimized or improved by the addition of Tob in Van/Tob-P.

Compressive Modulus (MPa)
4000 n=3
3500
3000
2500 *
2000 ** n=3
*
1500
1000 **
500
0 **
P-DCPD Van-P Van/Tob-P EM-P Tob-P
Figure 2. Compressive modulus of antibiotic-loaded P-DCDP cement scaffolds. Asterisk (*)
** cement. Error bars represent
denotes statistically significant difference (p < 0.05) to drug-free
SD.
16
70 n=3
Ultimate Compressive Strength

60
50
40
(MPa)
30 *
20
10
0
P-DCPD Van-P Van/Tob-P EM-P Tob-P
Figure 3. Compressive strength of antibiotic-loaded P-DCDP cement scaffolds. Asterisk (*)

denotes statistically significant difference (p < 0.05) to drug-free cement. Error bars represent
SD.
2.3.5 Cement Degradation
30 n=3
Figure 4. Degradation of EM-loaded P-DCDP cement scaffolds. Asterisk * (*) denotes

25
statistically significant difference (p < 0.05) to drug-free cement. Error bars represent
SD.Figure 3. Compressive strength of antibiotic-loaded P-DCDP cement scaffolds. Asterisk
(*) denotes
20 statistically significant difference (p < 0.05) to drug-free cement. Error bars
% Mass Loss
represent SD.
15
10
0
P-DCPD EM-P
Figure 4. Degradation of EM-loaded P-DCDP cement scaffolds. Asterisk (*) denotes

As shown in Fig. 4, embedding of EM at current concentration (10%) significantly
increased the cement degradation (24.97 %) as compared to P-DCPD (15.54%, p<0.05) and all
other groups.
17
25 n=3
20
% Mass Loss
15
*
10
0
P-DCPD Van/Tob-P Van-P Tob-P
Figure 5. Degradation of antibiotic-loaded P-DCDP cement scaffolds. Asterisk (*) denotes

Figure 6. In vitro degradation of antibiotic-loaded P-DCDP cement scaffolds.Figure 5.

Degradation of antibiotic-loaded P-DCDP cement scaffolds. Asterisk (*) denotes
Figure 6. In vitro degradation of antibiotic-loaded P-DCDP cement scaffolds.

18
As shown in Fig. 5, embedding of Tob at current concentration (10%) significantly slow
sdown the cement degradation (9.83%) as compared to P-DCPD (15.54%, p<0.05), and Tob-P
discs were visibly less degraded than other cement groups by the end of the four-week immersion
period (Fig. 6). Embedding of either Van or both Van/Tob slightly increased the cement
degradation (17.67% and 18.33%. respectively) but no statistical significance was found (Fig.5).
2.3.4 Cumulative Drug Release and Drug Concentration
Cumulative Drug Release n=3
100 *
*
90
% Drug Release
80
70
60
50
40 EM
30
20 *
10 *
0 *
0 5 10 15 20 25
Time (d)
Figure 7. Cumulative drug release of EM-doped P-DCPD cement. Asterisk (*) denotes
represent SD.
100
Figure 7.90Cumulative drug release of EM-doped P-DCPD cement. Asterisk (*) denotes
% Drug Relesed
statistically
80 significant difference (p < 0.05) between EM and all other groups. Error bars
represent70 SD.
60
50
40 n=3
EM
30 *
20 *
10 *
0
0 24 48 72
Time (h)
Figure 8. Cumulative drug release of EM-doped P-DCPD cement for first 72 hours.
Asterisk (*) denotes statistically significant difference (p < 0.05) between EM and all other
groups. Error bars represent SD.
Figure 8. Cumulative drug release of EM-doped P-DCPD cement for first 72 hours.
19
Drug Concentration n=3
5000
Drug Concentration (ug/ml)

4000
3000
*
2000 EM-P
1000
0
0 5 10 15 20 25
Time (d)
represent SD.

statistically
5000significant difference (p < 0.05) between EM and all other groups. Error bars
represent SD.
4500
Concentration (ug/ml)
4000
3500
3000
Figure 10.
2500Eluent drug concentration
* of EM-doped P-DCPD cement for the first 72 hours.
2000 EM-P
groups. Error bars represent SD.Figure 9. Eluent drug concentration of EM-doped P-DCPD
cement.1500
Asterisk (*) denotes statistically significant difference (p < 0.05) between EM and
all other1000
500
0
0 20 40 60
Figure 3. Eluent drug concentration of EM-doped
Time (h) P-DCPD cement. Asterisk (*) denotes
represent SD.
Figure 10. Eluent drug concentration of EM-doped P-DCPD cement for the first 72 hours.
No burst release of EM was observed, and >90% of EM was released after 28 days, as
shown in Fig.Figure 11. Cumulative drug release of antibiotic-doped P-DCPD cement. Asterisk (*)
7. Roughly 30% of EM was released within 72 hours (Fig. 8), followed by a
denotes statistically significant difference (p < 0.05) to Van-P. Error bars represent
SD.Figure 10. Eluent drug concentration of EM-doped P-DCPD cement for the first 72
sustained and hours.
zero Asterisk
order release ofstatistically
(*) denotes EM. Drug concentration
significant difference (p measurements
< 0.05) between EMover
and time show that
all other groups. Error bars represent SD.
EM concentration in solution is considerably higher than reported MIC values for S. aureus (≥8
μg/ml) [ 19] from the initial timepoint to the end of the immersion period (Fig. 9 and 10).
20

* *
100 **
% Drug Release
90 *
80
70 *
60 Van
50 *
Tob
40
30 Van (Van/Tob)
20
Tob (Van/Tob)
10
0
0 10 20
Time (d)
Figure 11. Cumulative drug release of antibiotic-doped P-DCPD cement. Asterisk (*)
denotes statistically significant difference (p < 0.05) to Van-P. Error bars represent SD.
Figure 11. Cumulative drug release of antibiotic-doped P-DCPD cement. Asterisk (*)
(p < 0.05) to Van-P. Error bars represent n
Drug Concentration
denotes statistically significant difference =3
SD.
5000
*
4000
3000
*
* * nVan
=3
2000 *
* Tob
* * *
1000
* *
0 *
0F 5 10 15 20 * 25
i* *
Time (d)
g
u
Figure 12. Eluent drug concentration of Van and Tob-doped P-DCPD cement.
r
Asterisk (*) denotes statistically significant difference (p < 0.05) between groups.
e
Error bars represent SD.
1
2
.
E
l
u
e
n
t
d
r
u
g
c
o
n
c
e
n
21
Drug Concentration
5000 * n=3
4500
Concentration (ug/ml)
4000
3500
3000
2500 *
2000 Van
*
1500 * Tob
*
1000
500
0
0 20 40 60
Time (h)
Figure 13. Eluent drug concentration of Van and Tob-doped P-DCPD cement for the first
72 hours. Asterisk (*) denotes statistically significant difference (p < 0.05) between groups

Figure 4. Eluent drug concentration of Van and Tob-doped P-DCPD cement for the first 72
*
5000 (*) denotes statistically significant difference (p < 0.05) between groups
hours. Asterisk
4000
*
3000 *
Figure 13. Eluent drug concentration of Van and Tob-doped P-DCPD cement Van for thenfirst
=3
(Van/Tob)
2000
72 hours. Asterisk (*) denotes statistically significant difference (p < 0.05) between groups
* SD.
Error bars represent Tob (Van/Tob)
1000 * *
0
0 Fdrug concentration
Figure 5. Eluent 10 20Tob-doped P-DCPD cement for the first 72
of Van and
i * denotes statistically
hours. Asterisk (*) Time significant
(d) difference (p < 0.05) between groups
Error bars represent
g SD.
u eluent drug concentration of Van/Tob-doped P-DCPD cement.
Figure 14. Individual
Asterisk (*) denotes
r statistically significant difference (p < 0.05) between groups. Error bars
represent SD.
e
1
As shown in Fig. 11, a significant reduction of burst release of Van was observed. About
4
.
30% of Van was released within 72 hours (Fig. 13) followed by a sustained release where roughly
Figure 15. µ-CT Iscans of antibiotic-loaded P-DCPD cement discs.Figure 14. Individual
n
eluent drug concentration of Van/Tob-doped P-DCPD cement. Asterisk (*) denotes
60% of Van was released
statistically up todifference
significant day 28 (p (Fig. 11).
< 0.05) Likegroups.
between EM-P, thebars
Error eluted drug
represent SD. concentration from
d
i
Van-P was at all time points v
considerably higher than the reported MIC value for S. aureus strains
i
(≥8 μg/ml) [20] (Fig. 12,dFig. 13), however, Van concentration is still roughly half than EM
u
concentration at most timepointsa (Fig. 9). Less than 5% of Tob was released from Tob-P up to 28
l
e
l
u
e
n
t
22
days (Fig. 11), and there was a drastic difference in drug release % and concentration between
Van-P and Tob-P (Fig 12).
The release pattern of Van and Tob from Van/Tob-P was different than their release from
Van-P and Tob-P (Fig. 11). In the presence of Tob, a dramatic increase in burst release of Van was
observed and >90% was released within 72 hours and all of Van was released by day 14 (Fig. 11).
Tob release from Van/Tob-P was greater (<5%) than Tob release from Tob-P (<2%) (Fig. 11).
However, eluted Tob concentration at most timepoints from both Tob-P and Van/Tob-P were not
considerably higher than reported MIC values for S. aureus (5 μg/ml) [21] (Fig. 13, Fig. 14)
2.4 Discussion
In the first set of experiments, we aimed to characterize the effect antibiotic addition on the
physicochemical properties of P-DCPD. The setting time and injectability varied for all antibiotic
groups with respect to drug-free cement, but the most significant changes were the increase in
setting time observed in Tob-P and EM-P. This behavior is in accordance to the previous reports
where, in general, the addition of antibiotics tends to increase the setting time and reduce the
mechanical strength of cements [22, 23]. It has been established that the setting reaction of CPC
can be affected by the introduction of drugs to either the powder or liquid phase, where it can
manifest as changes in the crystal structure of the drug-free cement [24, 24]; Gbureck, Probst and
Thull noted that drug-free cement matrix consisted of needle like crystals, whereas antibiotic-
loaded cements displayed a cluster-like orientation in its crystal matrix in addition to extended
setting time [22, 24]. In particular, it is possible that the increased setting time is due to an
inhibitory effect in the crystal formation during the dissolution and precipitation process of the
cement [24]. While a small percentage of antibiotic concentration may only exhibit negligible
effects in cement performance, it has been reported that this possible setting inhibitory effect tends
23
to become more prominent with increments in antibiotic concentration [24]. In addition, these
changes in setting time can also manifest as changes in the rheological properties of the cement
paste; Gbureck, Probst and Thull mention liquefication and increased workability of the cement
paste after the addition of gentamycin [24], whereas in our experiments it can be seen that the
drastic increase in setting time for cement doped with tobramycin, a hydrophilic drug, also brought
improved injectability compared to other antibiotic-loaded cement groups. However, specific
interactions between the antibiotic and the cement matrix must be considered, as the injectability
of cement doped with erythromycin, a hydrophobic drug, was not improved despite its increased
setting time.
The zeta potential of a surface gives us insight regarding the electrostatic stabilization of
particles in liquid phase and subsequent behaviors like agglomerization or adsorption, as well as
an idea regarding the rheological properties of powder suspensions. [24]. A zeta potential with a
magnitude larger than 25 mV suggests a highly charged surface and stability of colloidal particles
in solution [25]. Therefore, the larger zeta potential magnitude of Van-P than that of P-DCPD
indicates a higher stability, which could be attributed to the electrostatic repulsion among particles.
On the other hand, Tob-P and Van/Tob-P have a lower magnitude, which indicates less adsorption
of cations like calcium ions on the cement surface.
As previously mentioned, the addition of antibiotics tends to reduce the mechanical
strength of doped cements [24]. Specifically, the addition of drugs may precipitate an increment
in cement porosity, which is known to drastically reduce mechanical strength [26]. While EM-P
and Tob-P showed improved mechanical properties with respect to P-DCPD, the addition of
vancomycin considerably weakened P-DCPD cement scaffolds. µ-CT scans of the cement discs
allow the qualitative assessment of disc structure, and it seems that while Tob-P discs have a
24
smoother surface and denser appearance, Van-P discs have a more irregular surface with larger
pores (Fig. 15). Other than a presumed increase in the porosity of Van-P, a possible explanation
for the reduction in compressive strength and compressive modulus may relate to the presence of
halogenous ions in the vancomycin hydrochloride, the specific drug with which the cement was
doped with. Ratier et al have reported weak mechanical properties of CPC loaded with tetracycline
hydrochloride, and suggest that chloride ions have a strong affinity for adsorption on calcium
phosphates that inhibits the formation of intermediate phases of the cement and ultimately weaken
the overall cement structure [27]. Thus, the presence of chloride ions may be a more disruptive
factor in the crystallization of the cement matrix than drug polarity with respect to the cement’s
liquid phase alone.
Figure 15. µ-CT scans of antibiotic-loaded P-DCPD cement discs.

It must also be noted that the stress-strain curves for drug-free and drug-loaded cements
present a toe-region not typically observed in inorganic materials like calcium phosphate cements;
however, we attribute this toe-region to the slightly uneven surface of the cement scaffolds which
may have caused the scaffolds to initially buckle in an irregular manner.
Degradation of DCPD cements in vitro can occur via dissolution, disintegration or
conversion of DCPD to a more basic species like HA. [28]. In particular, dissolution takes place
when DCPD is placed in a solution undersaturated in phosphate and calcium ions, which is what
25
results in cement mass loss. [28]. In turn, high degrees of dissolution lead to microstructural
changes in the cement that result in disintegration and further increases mass loss [28]. Dissolution
can only take place as long as the solution is not saturated with respect to DCPD. [28]. We found
that, compared to P-DCPD, EM-P had a significantly higher mass loss percentage while Tob-P
had a significantly lower mass loss percentage. It may be that, considering the polarity of either
antibiotic, specific drug interactions with the cement matrix as well as the medium in which the
samples were submerged could affect the dissolution of ions and therefore the resulting loss of
mass.
For any drug delivery device, drug release depends on the device’s microstructure, drug
solubility and the bond between the drug and the device matrix, as well as the mechanism and rate
of degradation of the device, if present [22]. Being that CPC matrix is formed by a network of
interlocking crystals, the cement’s structure is very porous, displaying open micro or nanoporosity
[22]. Furthermore, as previously mentioned, DCPD is known to have a much higher degradation
rate than HA cements, so changes in pore size during drug release may play a major role in the
kinetics of drug release [22]. In the case of our drug-loaded P-DCPD cements, and especially in
the instance of poorly water-soluble drugs like EM, the release is proportional to time and
controlled by matrix dissolution [22]. Although drug diffusion is an important mechanism
controlling drug liberation and can be effectively observed for all groups when in contact with a
solid medium like agar, dissolution is the main mechanism of drug release in solution for P-DCPD
cement. Therefore, in accordance to the disc degradation results, EM-P discs released more than
95% of EM in 28 days, whereas Tob-P discs did not exceed 5% release of Tob within the same
period of time. Interestingly, Van-P discs released approximately 76% of its drug content
throughout 28 days with a slightly more pronounced initial burst than that of EM-P, but Van/Tob-
26
P exhibited a much more drastic Van burst release and all of the Van within the disc was released
by day 14, implying that the presence of Tob could have a negative effect on the binding
interactions between Van and the cement matrix.

27
CHAPTER 3 – ANTIBACTERIAL EFFICACY OF ANTIBIOTIC-LOADED P-DCPD

CEMENT
3.1 Introduction
Staphylococcus aureus (S. aureus) is responsible for up to 90% of the cases of pyogenic
osteomyelitis, while Staphylococcus epidermidis (S. epidermidis) seems to mainly infect medical
devices like orthopedic hardware implants and catheters [3]. In addition to a thick cell wall, around
80 to 90% of S. aureus strains possess a protective capsule that provides the bacterium with
antiphagocytic properties by preventing the host to recognize the invading microorganism [4]. S.
aureus is especially threatening during bone infections due to its propensity to form biofilms and
difficulty to eradicate without prolonged treatment [4]. We assessed the drug-loaded P-DCPD
cements antibacterial efficacy via direct contact on agar plate through three different ways:
antibiotic-loaded P-DCPD cement discs, drug eluent-soaked paper discs, and P-DCPD cement
discs prepared with post-degradation powder. In this way, antibacterial efficacy could be assessed
taking into account diffusion and degradation drug release mechanisms.
3.2.1 Bacterial Culture
Planktonic cultures of Staphylococcus aureus (Xn29 strain) were grown overnight in Luria
Bertani broth (LB, Thermo Fisher Scientific, Waltham, MA) at 37 °C. Cultures were then diluted
in fresh media to an optical density (determined using a spectrophotometer at OD 600 nm)
corresponding to approximately 1.6 x e8 CFU ml−1 to constitute a modified MIC assay.
3.2.2 Modified antibiotic minimum inhibitory concentration (MIC) assay
Agar diffusion assays were used to determine the release and potency of antibiotics from
drug-loaded P-DCDP cement discs over time. A bacterial lawn was prepared on LB agar plates
28
using 50 µl of planktonic culture Xn29 S. aureus. P-DCPD cement discs were transferred onto the
plates with sterile forceps and the plates incubated for 24 h at 37 °C. Zones of inhibition (ZOI)
were photographed and the discs were removed onto a freshly prepared lawn of bacteria every
other day until the ZOI was lost. The ZOI (radius, mm) was calculated using Image J (version
1.48) with the known petri dish diameter and metric ruler used for spatial calibration. All groups
were tested in duplicate.
3.2.3 Statistical Analysis
The software Microsoft Excel was used in the statistical analysis. Mean and standard
deviation were calculated from the experiment results. One-way ANOVA was used to analyze the
difference between group ZOI means. The statistical significance (p) < 0.05.
3.3 Results
3.3.1 Drug-loaded P-DCPD Cement Discs
Day 1 Day 4
Day 6 Day 11
Day 14 Day 16
29
Figure 16. Antibiotic-loaded P-DCPD cement disc time course.
P-DCPD Cement Disc n=3
14
12 *
*
10 *
ZOI (mm)
*
8
6 EM-P
4
2
0
0 5 10 15 20
Time (d)
Figure 17. EM-doped P-DCPD cement ZOI. Asterisk (*) denotes statistically
significant difference (p < 0.05) between EM and all other groups. Error bars
represent SD. Error bars represent SD.
30
P-DCPD Cement Disc n=3
14
12
10
ZOI (mm)
*
8 *
* Van-P
6 Van/Tob-P
4 *
Tob-P
2
0
0 5 10 15 20
Time (d)
Figure 18. Antibiotic-doped P-DCPD cements ZOI. Asterisk (*) denotes

statistically significant difference (p < 0.05) to Tob. Error bars represent SD.
Fig. 16 shows the agar ZOI for drug-loaded P-DCPD cement discs over a 20-day period.
Throughout the 20-day course, EM-P discs were markedly superior in the inhibition of bacterial
Figure 19. Drug eluent-soaked paper discs for bacterial inhibition assay.Figure 18.
growth, with statistically significant
Antibiotic-doped P-DCPDdifferences
cements ZOI.toAsterisk
other groups in statistically
(*) denotes ZOI radius for the last four
significant difference (p < 0.05) to Tob. Error bars represent SD.
timepoints (Fig. 17). Van-P discs showed an increased ZOI at the beginning but ultimately had
comparable ZOI radii to Tob-P and Van/Tob-P discs (Fig. 18).
3.3.2 Drug Eluent
Figure 19. Drug eluent-soaked paper discs for bacterial inhibition assay.
Figure 20. EM-doped P-DCPD eluent ZOI.Figure 19. Drug eluent-soaked paper discs for bacterial inhibition assay.
31
Eluent n=1
14
12
10
ZOI (mm)
8
6
4
2
0
0 5 10 15 20 25
Time (d)
Figure 20. EM-doped P-DCPD eluent ZOI.
Eluent n=1
Figure 6. EM-doped P-DCPD eluent ZOI.
10
9
8
Figure 21.
7 Antibiotic-doped P-DCPD eluent ZOI.Figure 20. EM-doped P-DCPD
ZOI (mm)
eluent ZOI.
6
5 Van-P
4 Tob-P
3
Figure 7. EM-doped P-DCPD eluent ZOI. Van/Tob-P
2
1
0
0 10 20
Time (d)
Figure 21. Antibiotic-doped P-DCPD eluent ZOI.
Fig. 19 shows agar ZOI for paper discs soaked in drug-loaded P-DCPD cement disc eluent at
every collected timepoint over a 28-day period. Following a similar pattern to that of drug-loaded
Figure 22. Degraded P-DCPD powder discs for bacterial inhibition assay.Figure 21.
P-DCDP cementAntibiotic-doped
discs, EM-P eluent
P-DCPDshowed the largest and most sustained ZOI (Fig. 20), followed
eluent ZOI.
by Van-P eluent (Fig. 21). The bacterial inhibition from Van/Tob-P eluent had an initial ZOI
comparable to that of Van-P but saw a sharp decrease over time, and the bacterial inhibition from
Tob-P eluent was negligible (Fig. 21).

32
3.3.3 Degraded P-DCPD Powder Discs
Figure 22. Degraded P-DCPD powder discs for

bacterial inhibition assay.
n=2
Degraded Powder Disc
Figure 8. Degraded P-DCPD powder discs for
8.00 * inhibition assay.
bacterial
7.00
6.00
Figure 23. Antibiotic-doped degraded P-DCPD
ZOI (mm)
5.00
powder discs ZOI. Asterisk (*) denotes
4.00 statistically significant difference (p < 0.05) to all
other groups. Error bars represent SD.Figure 22.
3.00 Degraded P-DCPD powder discs for bacterial
inhibition assay.
2.00
1.00
0.00 Figure 9. Degraded P-DCPD powder discs for
EM-P inhibition Van-P
bacterial assay. Tob-P Van/Tob-P
Figure 23. Antibiotic-doped degraded P-DCPD powder discs ZOI. Asterisk (*)
denotes statistically significant difference (p < 0.05) to all other groups. Error bars
represent SD.
Fig. 22 shows agar ZOI for discs pressed from drug-loaded P-DCPD powder recovered from
the 28-day degradation test. In accordance to bacterial inhibition results from drug-loaded cement
Figure 24. Impact of EM-doped P-DCPD eluent on cell proliferation. Asterisk (*)
denotes statistically significant difference (p < 0.05) to drug-free P-DCPD eluent.
discs and eluent,Error
EM-P produced
bars represent a statistically
SD.Figure significantly
23. Antibiotic-doped degradedlarger
P-DCPD ZOI than
powder other
discs groups (Fig.
ZOI. Asterisk (*) denotes statistically significant difference (p < 0.05) to all other
23). groups. Error bars represent SD.
33
3.4 Discussion
We tested the antibacterial efficacy of intact (non-degraded) cement discs by direct contact
with agar plate for zone inhibition assay. Bacterial growth was inhibited by both contact with drug
molecules exposed to the scaffold surface and the drug released from the scaffold matrix. EM-P
showed the strongest and most consistent zone inhibition throughout the entire study time frame
(20 days). We propose that the zone inhibition is expected to last longer, until the embedded EM
is completely released. The other drug-loaded cement groups showed strong zone inhibition on the
first two days. Both Van-P and Van/Tob-P maintained their bacterial inhibition activity for > 2
weeks. However, Van-P showed much strong zone inhibition than that of Van/Tob-P starting at
day 4 till 14 days (p<0.05). The zone inhibition activity from Tob-P was very limited within the
first 3 days, in part due to their very limited release and also to the slower degradation in dried
condition.
Furthermore, as shown in Fig. 19, eluents collected at given times showed a constant and
strong inhibitory effects of bacterial growth zone inhibition 28 day and up. This finding agrees
with what we observed for the EM release profile that indicated the EM was completely released
throughout 28 days. Eluents collected from both Van-P and Van/Tob-P showed similar zone
inhibition within the first 72 hours, although eluents from Van-P showed a much longer bacterial
growth inhibition up to 28 days than that of Van/Tob-P (p<0.05). Eluents from Van/Tob-P lost
their zone inhibition completely after 14 days. This finding agreed with what we observed for the
drug release profile that indicated the Van was completely released within 14 days. It also indicates
that the zone inhibition by eluents from Van/Tob-P is mainly caused by the Van in the eluents. In
agreement with the release profile of Tob in Fig. 11 and 12, a very limited release of Tob in the
eluents at all given times was not sufficient to inhibit bacterial growth.
34
To answer the question regarding if the degraded drug-loaded cements still retain some
bactericidal activity, we repeated the zone inhibition assay using the residue of cements after drug
elution up to 28 days. As shown in Fig. 22, the residues of degraded EM-P retain their strong zone
inhibition activity. We propose that zone inhibition does not entirely rely on drug released from
the cement disc but mainly on the contact surface on degrading scaffold surface via diffusion. This
further confirms our hypothesis that the bactericidal efficacy does not only depend on the amount
and rate of drug released, but also on the direct surface to surface contact with cells and bacteria.
Therefore, the degrading cements are expected to keep the drug efficacy until being completely
degraded.
35
CHAPTER 4 – CYTOCOMPATIBILITY OF ANTIBIOTIC-LOADED P-DCDP

CEMENTS
4.1 Introduction
The cytocompatibility of antibiotic-loaded P-DCPD cements was evaluated against
MC3T3-E1 murine pre-osteoblasts, which originate from C57BL/6 mouse calvaria. We assessed
the cytotoxicity of drug-loaded cement particles, drug-loaded cement particles after the 28-day
degradation period and drug release eluent at different concentrations, as well as the particles and
eluents impact on the cells’ ability to proliferate using LDH and MTT tests, respectively.
4.2.1 Cell Seeding and Culture
MC3T3 cells (ATCC, Manassas, VA) were cultured in a-modified minimum essential
medium (a-MEM, Invitrogen) supplemented with 10% fetal bovine serum (FBS, Invitrogen), 10
mM b-glycerophosphate (Sigma Aldrich), and 1% (v/v) mixture of penicillin and streptomycin
(Invitrogen). To assess the impact of direct contact with P-DCPD particles on cell growth and
cytotoxicity, particles were added to cell culture wells at different doses (0.1 µg/ml, 10/ml µg and
100 µg/ml, respectively). P-DCPD particles were sieved and inspected under optical microscope
to ensure <15 um particle size and UV-sterilized overnight prior to use. To assess the impact of
P-DCPD cements through indirect contact, drug release eluent from given timepoint was added to
cell culture wells at 10% v/v concentration. Cells cultured in the absence of cement particles or
eluent were included as controls and all groups were cultured in triplicate for three days.
4.2.2 Cell Proliferation Assay (MTT)
Cell proliferation was evaluated measuring the reduction of tetrazolium salts, which is
reduced by metabolically active cells. The resulting intracellular purple formazan can be
36
solubilized and quantified as a colorimetric assay following the manufacturer’s instructions
(ATCC, Manassas, VA). Fresh culture medium was used as a blank, and proliferation was
expressed as absorbance (OD).
4.2.3 Cell Toxicity Assay (LDH)
Cell toxicity was determined by measuring the release of LDH from dead or dying cells
into the culture medium by colorimetric method following the manufacturer’s instruction (Roche
Diagnostics BmbH, Indianapolis, IN). Fresh culture medium was used as a blank, and the total
cell lysate was used as a positive control. Cytotoxic activity was expressed as absorbance (OD).
4.3 Results
4.3.1 Cell Proliferation (MTT)
Eluent n=3
0.9
0.8
0.7
0.6
0.5 * *
OD
* * P-DCPD
0.4 * * *
* * EM-P
0.3
0.2
0.1
0
2 h 6 h 1 d 2 d 3 d 7 d 14 d 21 d 28 d
Figure 24. Impact of EM-doped P-DCPD eluent on cell proliferation. Asterisk (*)
37
P-DCPD Particle n=3
0.7
0.6 *
0.5
0.4
OD
0.3
0.2
0.1
0
Control P-DCPD EM-P 1 EM-P 10 EM-P 100
Figure 25. Impact of EM-doped P-DCPD particles on cell proliferation. Asterisk

(*) denotes statistically significant difference (p < 0.05) to drug-free P-DCPD
particles. Error bars represent SD.
Figure 26. Impact of degraded EM-doped P-DCPD particles on cell proliferation.

Line over bars denotesDegraded
no statisticallyP-DCPD
significant Particle n=3
difference between groups.
Error bars represent SD.Figure 25. Impact of EM-doped P-DCPD particles on cell
0.7 Asterisk (*) denotes statistically significant difference (p < 0.05) to
proliferation.
drug-free
0.6P-DCPD particles. Error bars represent SD.
0.5
0.4
OD
0.3
0.2
0.1
0
Control P-DCPD EM-P 1 EM-P 10 EM-P 100
Figure 26. Impact of degraded EM-doped P-DCPD particles on cell proliferation.
Line over bars denotes no statistically significant difference between groups.
Figure 27. Impact of antibiotic-doped P-DCPD eluent on cell proliferation. Line

over bars denotes no statistically significant difference between groups. Error bars
represent SD.Figure 26. Impact of degraded EM-doped P-DCPD particles on cell
proliferation. Line over bars denotes no statistically significant difference
between groups. Error bars represent SD.
38
Eluent n=3
0.9
0.8
0.7
0.6 P-DCPD
0.5
OD
Van-P
0.4
Tob-P
0.3
0.2 Van/Tob-P
0.1
0
2 h 6 h 1 d 2 d 3 d 7 d 14 d 21 d 28 d

represent SD.
P-DCPD Particles n=3

0.8
represent
0.6SD.
OD
0.4
0.2Impact of antibiotic-doped P-DCPD particles on cell proliferation.
Figure 28.
Line over0bars denotes no statistically significant difference between groups.
Error bars represent SD.Figure 27. Impact of antibiotic-doped P-DCPD eluent on
cell proliferation. Line over bars denotes no statistically significant difference
11. Impact of antibiotic-doped P-DCPD particles

Figure 28. eluent onon
cellcell
proliferation. Line
proliferation.
over
Line bars
over denotes no statistically
bars denotes significant
no statistically difference
significant between
difference groups.
between Error bars
groups.
represent
Error barsSD.
represent SD.
Degraded P-DCPD Particles n=3
0.7
0.6Impact of degraded antibiotic-doped P-DCPD particles on cell
Figure 29.
0.5 Line over bars denotes no statistically significant difference
proliferation.
between0.4
OD
groups. Error bars represent SD.Figure 28. Impact of antibiotic-doped P-

0.3
DCPD particles on cell proliferation. Line over bars denotes no statistically
0.2 difference between groups. Error bars represent SD.
significant
0.1
0
Figure 29. Impact of degraded antibiotic-doped P-DCPD particles on cell

proliferation. Line over bars denotes no statistically significant difference
Figure 30. Impact of EM-doped P-DCPD eluent on cell toxicity. Asterisk (*)
39
As shown in Fig. 24, the eluents from EM-P significantly inhibited cell growth at all time
points as compared to P-DCPD (p<0.05), while eluent from P-DCPD is safe and had no inhibitory
effects on cell growth. Embedding of EM-P particles only had a statistically significant effect on
viability at the highest concentration (Fig. 25). As shown in Fig. 27-29, the embedding of Van-P
and Tob-P particles and eluents did not inhibit the cell growth as compared to P-DCPD. Another
interesting question is whether degrading cement particles (28 days after drug eluting study and
majority of embedded antibiotics had been released) are still biocompatible and safe under the
same cell culture condition. As shown in Fig. 26 and 29, a similar cell growth pattern as observed
in non-degraded cements was found. Cement particles with or without antibiotics doping showed
the same proliferation pattern as the cement-free controls, even at higher concentration (100
µg/ml).
4.3.2 Cytotoxicity (LDH)
Eluent n=3
1.6 *
1.4
1.2
1
OD
0.8 Drug-free
*
0.6 * EM
0.4
*
0.2
0
2 h 6 h 1 d 2 d 3 d 7 d 14 d 21 d 28 d
Figure 30. Impact of EM-doped P-DCPD eluent on cell toxicity. Asterisk (*)
40
0.18
0.16
0.14
0.12
0.1
OD 0.08 *
0.06
0.04
0.02
0
Control Drug-free EM 1 ug/ml EM 10 EM 100
ug/ml ug/ml
Figure 31. Impact of EM-doped P-DCPD particles on cell toxicity. Asterisk (*)
denotes statistically significant difference (p < 0.05) to drug-free P-DCPD
Degraded P-DCPD Particle n=3

Figure 12. Impact of EM-doped P-DCPD particles on cell toxicity. Asterisk (*)
denotes0.25
statistically significant difference (p < 0.05) to drug-free P-DCPD
0.2
0.15
OD
Figure 32. Impact of degraded EM-doped P-DCPD particles on cell toxicity. Line
0.1
represent SD.Figure 31. Impact of EM-doped P-DCPD particles on cell toxicity.
0.05
Asterisk (*) denotes statistically significant difference (p < 0.05) to drug-free P-
DCPD particles.
0 Error bars represent SD.
Control Drug-free EM 1 ug/ml EM 10 EM 100
ug/ml ug/ml
Figure 13.
32. Impact of degraded
EM-dopedEM-doped
P-DCPD particles
P-DCPDon cell toxicity.
particles on cellAsterisk
toxicity.(*)
Line
denotes
over barsstatistically
denotes nosignificant
statisticallydifference (p difference
significant < 0.05) to between
drug-freegroups.
P-DCPD Error bars
particles.
represent Error
SD. bars represent SD.
Eluent n=3
over bars0.4
denotes no statistically significant difference between groups. Error bars
represent SD.
0.35 *
0.3 *
* Drug-free
0.25 *
OD
Figure 33.0.2
Impact of antibiotic-doped
* P-DCPD eluent on cell toxicity. Asterisk (*)
Van
0.15
Error bars represent SD.Figure 32. Impact of degraded EM-doped P-DCPD Tob
particles on
0.1cell toxicity. Line over bars denotes no statistically significant
Van/Tob
difference
0.05between groups. Error bars represent SD.
0
2 h 6 h 1 d 2 d 3 d 7 d 14 d 21 d 28 d
Figure 33. Impact of antibiotic-doped P-DCPD eluent on cell toxicity. Asterisk
(*) denotes statistically significant difference (p < 0.05) to drug-free P-DCPD
represent SD.
eluent. Error bars represent SD.
Figure 34. Impact of antibiotic-doped P-DCPD particles on cell toxicity. Line

41
0.2
0.16
0.12
OD
0.08
0.04
0
Figure 34. Impact of antibiotic-doped P-DCPD particles on cell toxicity. Line

represent SD.
Degraded P-DCPD
Figure 16. Impact of antibiotic-doped Particles
P-DCPD particles n=3
on cell toxicity. Line
represent0.3
SD. *
0.25
0.2
OD
0.15
0.1
0.05
0
Figure 35. Impact of degraded antibiotic-doped P-DCPD particles on cell

proliferation. Asterisk (*) denotes statistically significant difference (p < 0.05) to
degraded drug-free P-DCPD particles. Error bars represent SD.
Comparing EM-P eluent to P-DCPD eluent, a significant increase of LDH concentration
was only found in the eluent collected at 7, 14 and 21 days. (p<0.05). There is no significant
relationship between LDH concentration with the absolute EM concentration in the eluent samples
(Fig. 30). In agreement with the cell growth data, EM-P was not toxic as manifested by lower LDH
concentration than that of controls even at higher concentration (100 µg/ml) (Fig. 31, 32).
As compared to control, a slight reduction of LDH concentration was observed in all drug-
doped cement eluents. However, there is no significant statistical difference. Taken together, cells
42
exposed to the eluents at 10% had a lesser impact on cell growth and were not toxic to MC3T3
cells (Fig. 33). In agreement with the cell growth data, cement particles with or without drug
doping did not have cytotoxic impacts on cells as manifested by lower LDH concentration than
that of controls even at higher concentration (100 µg/ml) (Fig. 34, 35). We also noticed that cells
treated with lower concentration of antibiotics (1 µg/ml and 10 µg/ml particle) had much lower
LDH concentrations as compared to that of controls. (p<0.05).
However, treatment with cement particles from antibiotics doping group at highest
concentration (100 µg/ml) had a significant higher LDH concentration as compared to that of
cement free controls (p<0.05). However, no statistical difference was found among cement with
the type of antibiotics doped.
4.4 Discussion
The down regulation of cell growth by eluents from EM- cements could not be explained
by the cell toxic reaction. One possibility is that EM-P eluents stimulated cell differentiation
instead of cell growth that we reported before for the EM released from nanofiber materials [29],
where cell differentiation experiments were designed to test this hypothesis. EM-cement is
biocompatible and that is further supported by the direct contact culture models.
The same pattern of cell growth and LDH concentration was observed when cells exposed
to degrading EM-P (28 days after drug eluting study and >90% of embedded EM was released).
A similar cell growth pattern as observed in undegrading cements was found even at higher
concentration (100 µg/ml). However, treatment with EM-P particles at highest concentration (100
µg/ml) had a significant higher LDH concentration as compared to that of cement free controls
(p<0.05). Interestingly, a dose dependent reduction of LDH concentration was observed (p<0.05),
although the reason is not clear. We propose that calcium or phosphate ions and other components
43
released during cell culture (in addition to antibiotics released) might have some inhibitory effects
on LDH activity measurement.

44
CHAPTER 5 – CONCLUSION AND FUTURE WORK
In this study, the physicochemical characteristics and changes brought upon the addition
of antibiotics to P-DCPD cement matrices were assessed. We found that the addition of antibiotics
caused changes in cement setting time and injectability, often manifested as longer setting times
and most notably observed in the case of Tob-P and EM-P. Furthermore, the addition of drugs had
an impact on P-DCPD particles Zeta potential, particularly decreasing the magnitude of Tob-P and
Van/Tob-P Zeta potentials. While the addition of EM and Tob improved the mechanical properties
of P-DCPD cement, the addition of Van to the cement matrix caused a significant decrease in the
scaffolds’ compressive strength and compressive modulus. Regarding the impact of drug addition
to cement degradation rates, EM-P demonstrated a significant increase in mass loss percentage
over a four-week period while Tob-P degraded significantly less than drug-free cements. Similarly,
EM-P displayed sustained and efficient drug release, releasing more than 95% of its drug content
over a four-week period, while Tob-P did not dissolve enough to exceed 5% cumulative drug
release.
Regarding antibacterial efficacy, EM-P displayed significantly stronger and longer-lasting
bacterial inhibition than other drug-loaded cements across all three experimental setups. However,
EM-P had an overall negative impact on pre-osteoblastic proliferation compared to other groups,
although it did not exert a drastic cytotoxic effect on the cells.
In conclusion, although the addition of antibiotics to P-DCPD cement matrix can
significantly compromise the scaffold’s mechanical and handling properties, these effects vary in
intensity between different antibiotics. Taken together, drug-loaded P-DCPD cements, and in
particular those doped with EM, can act as effective carriers for sustained bacterial inhibition,
45
although the possibility of modulating the cement’s drug release to render it even slower could
reduce any drastic cytotoxic effects on native cell populations.
Future work for this project includes investigation of the drugs’ molecular interactions with
CPP gel and its effects on cement crystalline structure using Raman spectroscopy and XRD,
respectively. In addition, the quantification of cement porosity with respect to drug addition using
µCT scanning could provide useful data to further characterize the cement’s drug release kinetics.
Lastly, the assessment of these drug-loaded P-DCPD cements on biofilm inhibition and eradication
is of great interest and will be pursued as well.

46
APPENDIX: ABBREVIATIONS
ACP: amorphous calcium phosphate
ACPP: amorphous calcium polyphosphate (
CPC: calcium phosphate cement
CPP: calcium polyphosphate (
DCPA: dicalcium phosphate anhydrous
DCPD: dicalcium phosphate dihydrate
EM: erythromycin
HA: hydroxyapatite
MCPM: monocalcium phosphate monohydrate
MIC: minimum inhibitory concentration
MRSA: methicillin-resistant forms of Staphylococcus aureus
P-DCPD: poly-dicalcium phosphate dihydrate
PMMA: poly-methylmethacrylate
Tob: tobramycin
TTCP: tetracalcium phosphate
Van: vancomycin
ZOI: zone of inhibition
47
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51
ABSTRACT
RELEASE AND BIOACTIVITY OF ANTIBIOTICS FROM INJECTABLE

POLYMERIC DCPD CEMENT
by
ANGÉLICA GUARDIA
May 2020
Advisor: Dr. Weiping Ren
Major: Biomedical Engineering
Degree: Master of Science
Treatment of osteomyelitis remains a significant clinical challenge, with over 75% of cases
caused by commensal Staphylococcus aureus (S. aureus). Calcium phosphate cements (CPC) − in
particular, dicalcium phosphate dihydrate (DCPD) cements − have been proposed as an alternative
to polymethyl methacrylate (PMMA) cements as antibiotic delivery vehicles for the treatment of
contaminated bone defects due to their excellent osteoconductivity, similar mineral composition
to that of native bone tissue, and resorbability under physiological conditions. Unfortunately, there
is still no suitable solution to the problems of poor handling properties, low anti-washout
resistance, weak mechanical properties and burst drug release.
Polymeric DCPD (P-DCPD) is a new, injectable CPC obtained by the setting of calcium
polyphosphate (CPP) gel with tetracalcium phosphate (TTCP). P-DCPD represents a promising
bone cement alternative due to its strong mechanical properties, excellent anti-washout resistance
and controllable and sustained release of embedded drugs.
The purpose of this study is to assess the physicochemical properties of P-DCPD cement
loaded with erythromycin, vancomycin or tobramycin, as well as evaluate its bactericidal efficacy
against S. aureus and cytocompatibility with MC3T3 murine pre-osteoblasts at a 10% w/w drug-
52
loading concentration. The hypothesis is that the addition of antibiotics to P-DCPD cement matrix
will not significantly compromise the mechanical and handling properties of the cement while
inhibiting bacterial growth without exerting a drastic cytotoxic effect on pre-osteoblastic cells.
53
AUTOBIOGRAPHICAL STATEMENT
EDUCATION
Graduate Education (MS)
Master of Science Biomedical Engineering, Wayne State University, Department of Biomedical
Engineering. College of Engineering: Dr. Weiping Ren
Undergraduate Education (BS)
Bachelor of Science – Biomedical Engineering, Lawrence Technological University, Southfield,
MI.

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RELEASE AND BIOACTIVITY OF ANTIBIOTICS FROM INJECTABLE

POLYMERIC DCPD CEMENT

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LIST OF TABLES .................................................................................................................................... vi

LIST OF FIGURES ................................................................................................................................. vii

CHAPTER 1 – GENERAL INTRODUCTION ..................................................................................... 1

1.1 Brief Overview of Bone Anatomy and Physiology .............................................................. 1

1.2 Pathophysiology of Osteomyelitis ........................................................................................ 1

1.3 Antibiotic-loaded Bone Cements as Osteomyelitis Treatment ............................................. 3

CHAPTER 2 – PHYSICOCHEMICAL CHARACTERIZATION OF ANTIBIOTIC-LOADED

2.1 Introduction ........................................................................................................................... 5

2.1.2 Advantages of P-DCPD Cement .................................................................................... 7

2.2 Materials and Methods .......................................................................................................... 9

2.2.1 Preparation of CPP powder and ACPP hydrogel ........................................................... 9

2.2.2. Preparation of TTCP powder ......................................................................................... 9

2.2.3 Preparation of Drug-loaded P-DCPD Cement Samples ............................................... 10

2.2.3 Setting Time and Injectability ...................................................................................... 10

2.2.4 Zeta Potential ................................................................................................................ 10

2.2.5 Mechanical Testing (Compressive Modulus and Compressive Strength) .................... 10

2.2.6 Cement Degradation ..................................................................................................... 10

2.2.7 Cumulative Drug Release and Drug Concentration ..................................................... 11

2.3 Results ................................................................................................................................. 12

2.3.1 Setting Time and Injectability ...................................................................................... 12

2.3.2 Zeta Potential ................................................................................................................ 13

2.3.3 Mechanical Testing (Compressive Modulus and Compressive Strength) .................... 14

2.3.5 Cement Degradation ..................................................................................................... 16

2.3.4 Cumulative Drug Release and Drug Concentration ..................................................... 18

2.4 Discussion ........................................................................................................................... 22

CHAPTER 3 – ANTIBACTERIAL EFFICACY OF ANTIBIOTIC-LOADED P-DCPD

3.1 Introduction ......................................................................................................................... 27

3.2 Materials and Methods ........................................................................................................ 27

3.2.1 Bacterial Culture ........................................................................................................... 27

3.2.2 Modified antibiotic minimum inhibitory concentration (MIC) assay .......................... 27

3.2.3 Statistical Analysis ....................................................................................................... 28

3.3 Results ................................................................................................................................. 28

3.3.1 Drug-loaded P-DCPD Cement Discs ........................................................................... 28

3.3.2 Drug Eluent................................................................................................................... 30

3.3.3 Degraded P-DCPD Powder Discs ................................................................................ 32

3.4 Discussion ........................................................................................................................... 33

CHAPTER 4 – CYTOCOMPATIBILITY OF ANTIBIOTIC-LOADED P-DCDP CEMENTS.. 35

4.1 Introduction ......................................................................................................................... 35

4.2.1 Cell Seeding and Culture .............................................................................................. 35

4.2.2 Cell Proliferation Assay (MTT) ................................................................................... 35

4.2.3 Cell Toxicity Assay (LDH) .......................................................................................... 36

4.3 Results ................................................................................................................................. 36

4.3.1 Cell Proliferation (MTT) .............................................................................................. 36

4.3.2 Cytotoxicity (LDH) ...................................................................................................... 39

4.4 Discussion ........................................................................................................................... 42

CHAPTER 5 – CONCLUSION AND FUTURE WORK .................................................................. 44

APPENDIX: ABBREVIATIONS .......................................................................................................... 46

AUTOBIOGRAPHICAL STATEMENT ............................................................................................. 53

Table 1. Setting time and injectability of EM-doped P-DCPD cement. ....................................... 12