PROJECT PROPOSAL
A Novel Approach to Design Silver
Hydroxyapatite - Polymer
Nanostructured Biomaterials as
Antibacterial Bone Implants
BY
DR. SANJANA KANKANE
1.
INTRODUCTION
Existing orthopedic implants typically consists of a single bioinert material such
as metals, ceramics or polymers, or of a relatively coarse combination of two or three
components. Although these synthetic materials provide an immediate solution for many
patients, their long-term performance is generally not satisfactory (1). This is often due
to rarely bear functionalities of these traditional implants that encourage communication
with their cellular environment limiting the potential for self repair, adaptation to
physiological conditions and tissue attachment and in growth. (2)
Bone is the most implanted tissue after blood and it is a specialized form of
connective tissue that provides a framework for the body. Although bone is considered
as the strongest tissue in the body, it often undergoes damage or defect, resulting from
traumatic or non-traumatic destruction (3).
The unusual combination of a hard inorganic material and an underlying elastic
hydrogel network gives bone unique mechanical properties such as low stiffness,
resistance to tensile and compressive forces and high fracture toughness (4) Common
bone substitution materials are autografts, allograft, xenografts and various synthetic
material like polymers, metallic materials, composites and bioceramics (5,6). However,
none of these materials provide a perfect solution for guided bone healing because of
their mechanical stability, long term in vivo biocompatibility and biodegradability (7).
Kowai et al (8) suggested that collagen promotes the proliferation and attachment
of host osteoblastic cells on octacalcium phosphate/collagen composite implants. The
composite is expected to have improved mechanical properties compared to near polymer
and better structural integrity and flexibility than brittle ceramics. In fact the combination
of ceramics and polymers could provide reinforced porous structures with enhanced
bioactivity and controlled resorption rates (9). In order to overcome these problems
various synthetic grafts have been developed to provide an alternative [10,11) The ideal
bone graft should be osteoconductive so that it allows rapid integration with the host
bone. Furthermore, it should be biodegradable at an ideal rate to be replaced by newly
formed natural bone, and ultimately osteoinductive to allow rapid new bone formation
[12,13].
HAP (Fig.1) is a bone mineral, which comprise 90% of the inorganic matrix of
bone and is osteoconductive (14) It is widely used in surface coating of joint
replacements, as it facilitates direct bone in growth to the prosthetic component. It has
also been used as a bone graft substitute but is poorly desorbed (compared to calcium
sulphate). (15)
Fig.1 Crystal structure of hydroxyapatite.
Calcium sulphate-hemi hydrate (2 CaSO4 H20), known as „gypsum‟ or plaster of
paris, and HA have pure osteoconductive qualities, thus creating a suitable environment
for new bone formation. The combination of calcium sulphate and HA enables rapid
resorption of calcium sulphate but leaves a coral matrix of porous HA (Fig 2). This
osteoconductive scaffold has a large surface area, which further enhances in growth and
formation of new bone (16).
When hydroxyapatite is combined with naturally occurring polymers like
chitosan, its cytotoxic effect is neutralized. Chitosan is an amino polysaccharide and
used as a binder in this composite – is also biocompatible and biodegradable (17). It also
has antimicrobial effects and osteoconductive properties. Collagen type hydroxyapatite
and I were found to enhance osteoblastic differentiation (18), but when combined
together they were shown to accelerate osteogenensis. In addition col-HA composites
proved to be fairly biocompatible both in humans and in animals (19).
When comparing ceramic scaffolds with ceramic composite scaffolds, it was
shown that col-HA composites performed well compared to single HA or TCP scaffolds
(20). The addition of collagen to a ceramic structure can provide many additional
advantages to surgical applications, shape control, spatial adaptation and defect wall
adhesion and the capability to favor clot formation and stabilization (21)
Bigi et al (22) crosslinked gelatin with genipin and suggested that genipin which
is by far less cytotoxic can be considered a valid alternative for crosslinking gelatin
biomaterials. Genipin was used to crosslink Chitosan/gelatin blends promoting the
formation of amide and tertiary amine bonds between the macromolecules and the
crosslinker.
In the present research proposal attempts will be made to fabricate a new
antibacterial hydroxyapatite impregnated collagen based composites and to test the
efficacy of antibacterial hydroxyapatite composites on rat calvaria defects for bone
regeneration and then will be examined radiographically and histologically.
2.
OBJECTIVES
Since bacterial infection is a rising complication following the wide use of
implants, there is considerable attention to prepare antibacterial hydroxyapatite composite
as bone substitutes. The study includes at achieving the following objectives.
(1)
Preparation of antimicrobial hydroxyapatite powder having good affinity for a
living body and strong antimicrobial property.
(2)
Fabrication of antibacterial impregnated hydroxyapatite polymer composites of
various chemical architectures by adopting conventional synthetic routes.
(3)
Structural, morphological and thermal characterization of antibacterial apatitepolymer nanocomposites
(4)
Evaluation of water sorption capacity, mechanical properties, in vitro blood
compatibility, and cytotoxicity of prepared nanocomposites.
(5)
To perform in vivo studies by implanting the biomaterial on rat calvaria defect for
bone regeneration and examine radiographically and histologically.
(6)
To observe the antibacterial effect of AgHA composites against Escherichia Coli
and Staphylococcus aureus by established methods.
3.
PROPOSED METHODOLOGY OF THE RESEARCH WORK
Materials:
In order to prepare antibacterial HAP impregnated polymer nanocomposites, a
variety of naturally occurring polymers such as gelatin (both A and B), collagen,
chitosan, alginate, etc. will be used, structures of which are shown in Fig. 1 and 2. Other
chemicals / reagents to be used include genipin (crosslinker of gelatin) ,hydroxyapatite,
calcium sulphate, plaster of Paris, etc. Silver salt solution will be used for preparing silver
nanoparticles. All reagents and chemicals will be of standard quality grade.
Methods:
(i) Preparation of antimicrobial Hydroxyapatite
Silver hydroxyapatites have been recognized for their antibacterial behavior and
are produced as given below;
1.0 kg of hydroxyapatite and 0.0016 g of silver nitrate are added to 10 L of distilled
water and stirred for 1 hour with heating. The products are fully washed with distilled
water, dried and crushed. Antimicrobial hydroxyapatitte powders containing 0.001%
silver are obtained.(23 ) The dissolving test is performed on this product and no
silver is dissolved. The antimicrobial activity test will be performed by using a
composition comprising 1% of said product and 88% of Hydroxyapatite.
Hydroxyapatite is used as a control.
(ii) Preparation of silver HAP polymer composites
In order to prepare the polymer composites a genipin crosslinker will be used. In
brief known amounts of natural polymer collagen are taken in the solution with
crosslinking agent and then calculated amount of hydroxyapatite and plaster of Paris
will be mixed thoroughly. Polymerization is allowed to take place for a known
period so as to get a slab of the composites.
Characterization:
For characterization purpose , FTIR, UV-VIS-spectroscopy, SEM, Particle size analysis ,
XRD , TGA and Mechanical testing techniques will be used .
Water sorption study/ swelling in biofluids:
For determining the amount of water, conventional gravimatric method will be adopted.
In-vitro blood compatibility:
In-vitro blood compatibility will be adjudged by Clot formation test, Haemolysis Assay,
Platelet adhesion test, Protein Absorption and study of Antibacterial activity as reported
elsewhere.
In vivo studies on animals:
This project aims to evaluate the performance of newly developed antibacterial
composite as a bone grafting material. The composites will be fabricated in a circular disc
and implanted in a predrilled hole in a rat‟s/rabbit‟s calvaria defect (24) After
implantation the regenerations of bone tissue into the disc bone graft and its integration
with the remaining material will be observed on days 15, 30, 60,and 90 postoperatively to
assess .the status of the implant.
4.
TIME SCHEDULE
Ist year
In the first year of the project tenure, a series of antibacterial macromolecular matrices
possessing water sorption capacity will be synthesized by conventional polymerization in
the presence of different preformed polymer like collagen, gelatin, chitosan, etc. In this
way, in the first year of the project major time shall be devoted to the material synthesis.
After preparation the composites will be studied for their water sorption capacity and
biodegradability. First few months of the year may also be used in setting up laboratory.
IInd year
In the second year of the project the prepared antibacterial composites will be
characterized by FTIR, ESEM, TEM, and XRD techniques. Thermal and mechanical
studies will be carried out to evaluate their thermal stability, phase transformation and
mechanical properties such as compressive strength, tensile strength, Young‟s modulus of
the composites In-vitro blood compatibility and antibacterial property will also be
investigated with composites of different compositions.
IIIrd year
In the IIIrd year of the project tenure in vivo studies on animals like rat/rabbit shall be
done. Bone regeneration and biodegradability of implanted bone substitute would be
noticed histologicaly and radiologically. This part of the work may take about ten months
and the remaining two months will be utilized in completing the experimental data and
preparing final progress report.
5.
EXPECTED DELIVERABLES / OUTCOME
By a judicious selection of preformed polymer, monomers and silver
hydroxyapatitte a variety of antibacterial nanocomposites may be prepared which could
offer unique combination of properties such as biocompatibility, hydrophilicity,
mechanical strength and antibacterial property. Such materials could prove to be not only
an alternate of bone but may also show better performance especially in post surgery
cases where chances of infections are much greater.
Thus the present research proposal may give rise to such specialty
nanocomposites materials, which may open up a new pace to material science for
orthopedic applications.
6.
SIGNIFICANCE OF THE EXPECTED OUTCOME WITH
RESPECT TO THE STATE OF THE ART IN THE FIELD
In the light of the significance of the problems pertaining to need for bone like
materials the end polymer composites emerging out of the present research proposal may
be quite fruitful and may lead to successful designing of silver hydroxyapatitte- polymer
nanocomposites with high performance application value.
References –
(1)
(2)
(3)
(4)
(5)
(6)
(7)
(8)
(9)
(10)
(11)
(12)
(13)
(14)
(15)
(16)
(17)
Ma P.X., Zhang R.Y., Xiao G.Z., Franceschi R., J. Biomed. Mater. Res., 54,
284-293, (2001).
William G., Adv. Eng. Mater., 1, 95-105, (1999).
Murugan R., Ramakrishnan S., ”Development of Cell Responsive Nanophase
Hydroxyapatite for Tissue Engineering”, American Journal of Biochemistry
and Technology, 3(3), 118-124, (2007).
Weiner S., Wagner H.D., Annu. Rev. Mater. Sci., 28, 271-298, (1998).
Dorozhkin S.Y., Epple M., “Biological and Medical Significance of Calcium
Phosphates”, Angew. Chem. Int. Ed. Engi., 41, 3130-46, (2002).
Tadic D., Epple M., “A thorough physiochemical characterization of 14
calcium phosphate based bone substitution materials in comparison to natural
bone”, Biomaterials, 25(6), 987-94, (2004).
Tadic T., Beckmann F., Schwarz K., Epple M., ”A novel method to produce
hydroxyapatite objects with interconnecting porosity that avoids sintering”,
Biomaterials, 25, 3335-3340, (2004).
Kawai T., Anada T., Honda Y., Kamakura S., Matsui K., Matsui A., Sasaki K.,
Morimoto S., Echigo S., Suzuki O., “Synthetic octacalcium phosphate augments
bone regeneration correlated with its content in collagen scaffold”, Tissue Eng.
Part A, (2008).
Wang M., “Developing bioactive composite material for tissue replacement”,
Biomaterials, 24, 2133-57, (2003).
Betz R.R., “Limitations of autograft and allograft: new synthetic solutions”,
Orthopedics, 25(5), S561–S570, (2002).
Gazdag A.R., Lane J.M., Glaser D., Forster R.A., “Alternatives to autogenous
bone graft: efficacy and indications”, Journal of the American Academy of
Orthopaedic Surgeons, 3(1), (1995).
Vaccaro A.R., “The role of the osteoconductive scaffold in synthetic bone
grafts”, Orthopedics, 25(5), S571–S578, (2002).
Damien C.J., Parsons J.R., “Bone-Graft and Bone-Graft substitutes – A review
of current technology and applications”, Journal of Applied Biomaterials, 2(3),
187–208, (1991).
Homes R.E., Wardrop R.W., Wolford I.M., “Hydroxyapatite as a bone graft
substitute in orthognathic surgery; histologic and hismometric findings”, J. oral
Maxillaofae. Sur., 46, 661-71, (1988).
Orore A., Hotta T., Kawashima H., Kondo N., Cu W., Kamura T., et. al.
“Comparison of hydroxyapatite and beta tricalcium phosphate as bone substitute
after excision of bone tumors”, J. Biomed. Mater. Res. B Appl. Biomater., 72,
94-101, (2005).
Schindler O. S., Cannon S. R., Blunn G. W. “Composite ceramic bone graft
substitute in the treatment of locally aggressive benign bone tumours”, Journal
of orthopedic surgery, 16(1), 66-74, (2008).
Krisanapiboon A., Buranapanitkit B., Oungb K., “A biocompatibility of
hydroxyapatite composite as a local drug delivery system”, J. orthopedic
surgery, 14(3), 315-18, (2006).
(18)
(19)
(20)
(21)
(22)
(23)
(24)
Xie J., Baumann M.J., McCube L.R., ”Osteoblasts respond to hydroxyapatite
surfaces with immediate changes in gene expression”, J. Biomed. Mater. Res.
A, 71, 108-117, (2004).
Scabba A., Trombelli L., “A comparative study on the use of alfa HA collagen
chondroitin sulphate biomaterialand a bovin-derived HA xenograft in the
treatment of deep intraosseous defects”, J. Clin. Periodonto , 31, 348-355,
(2004).
Wang X., Grogn S.P., Rieser F., Winkelmann V., Maquet V., Berge M.L.,
Mainil-Varlet P., “Tissue engineering of biphasic cartilage constructs using
various biodegradable scaffolds an in vitro study”, J. Biomaterials , 25, 36813688, (2004).
Wahl D.A., Czermuszka J.T., ”Collagen-hydroxyapatite composites for hard
tissue repair”, J. European Cells and Materials, 11 , 43-56, (2006).
Bigi A., Cojazzi G., Panzavolta S., Roveri N., Rubini K., “Stabilization of
gelatin films by crosslinking with genipin”, Biomaterials, 23, 4827-4832,
(2002).
Sakuma S., Atsumi K., Fujita K., “Antimicrobial hydroxyapatite powders and
methods for preparing them”, Patent 5009898, (1991).
Hsu F.Y., Tsai S.W., Lan C.W., Wang Y.J., “An in vivo study of a bone grafting
material consisting of hydroxiapatite and reconstituted collagen”, J. Mat. Sci.
Mat. In Med., 16(4), 341, (2005).