Lecture about Antimicrobial Osteoinductive Materials to Spinal Surgery

1. Biomaterials
with
Antimicrobial and Osteoinductive
Properties applied to
Spinal Surgery
GEORGE SAPKAS
PROFESSOR OF ORTHOPAEDICS
Metropolitan Hospital Athens
Spinal Disorders and
Musculo-sceletal Unit

2. In the past decade,
novel biomaterials,
bearing both
antibacterial and
osteoinductive
properties, have been
developed in order to
provide a viable
treatment option for
infected bone defects.
Li, X., et al, Clin. Orthop. Relat. Res., 2013
Zheng, Z., et al, Biomaterials 2010

3. Many novel antibacterial
drugs, such as:
nanosilver/silver
nanoparticles (AgNPs),
quanternized chitosan,
Copper
show a very
promising clinical
application potential
due to their good
biocompatibility and a
broad bactericidal
spectrum
Franci, G., et al Molecules 2015
Tan, H., et al Int. J. Mol. Sci., 2013
Copper

4. Antimicrobial Biomaterials

5. AgNPs

6. AgNPs are clusters of
silver atoms with
diameters ranging from
1 to 100 nm. AgNPs
have become highly
interesting for medical
applications because of
their:
antimicrobial,
anti-inflammatory,
biocompatible and
wound-healing-favoring
properties
Chaloupka, K., et al, Trends Biotechnol. 2010

7. The powerful antimicrobial
activities of AgNPs can be
attributed to the following
orchestrated mechanisms:
the damage of bacterial
membranes;
the inhibition of DNA
replications, protein
synthesis and enzymatic
activity
the alteration of cell
respiration
Franci, G. et al, Molecules 2015

8. AgNPs have also
another advantageous
property over
antibiotics:
an antiviral effect

9. Qaternised Chitosan

10. Chitosan, a naturally-
derived polycationic
polymer, has been
made into various
biomedical devices
due to its
proper
biodegradability,
good biocompatibility
and antimicrobial
activity
Rabea, E.I., et al, Biomacromolecules 2003

11. Quanternized
chitosan could also
effectively kill
antibiotic-resistant
bacteria, such as
Methicillin-resistant
Staphylococcus
aureus

12. The mechanisms of the
antibacterial activities of
quanternized chitosan are
still not totally clear.
One of the major
mechanisms is the
clectrostatic interaction
between the polycationic
groups of quanternized
chitosan
and the anionic components of
microorganisms.

13. Copper

14. The first reports of copper
were recorded in texts of the
Assyrians and Egyptians in
4000 BC .
In one of the oldest books of
Edwin Smith papyrus mentions
the use of copper for sterilizing
not only chest wounds but also
drinking water
(2600-2200p.Ch.)
Hippocrates (460-480 BC) in
his reports makes observations
about the beneficial
capabilities of copper in the
treatment of wounds

15. Copper ions, upon
contact with microbes,
generates electric
deregulation and
disorder the pump of
K – Na which is located
in the cell membrane .
This action creates
osmotic problems in the
cell, thereby killing the
microbes .

16. Mechanism of copper ions Action
SYTO-9 PI
Cu+/Cu2
+
Cu2+
Metallic Copper as an Antimicrobial Surface .Marc Solioz et al,*Appl Environ Microbiol. 2011 77(5):
1541–1547
Deregulation
and disorder
the pump of K
– Na

17. Growth factors

18. Many growth factors,
such as:
basic fibroblast growth
factor,
insulin-like growth factors
(IGFs),
vascular endothelial
growth factor,
are also capable of
significantly promoting
osteogenesis and
angiogenesis
Wang, J., et al Int. J. Mol. Sci. 2014

19. However, only
bone morphogenetic
proteins (BMPs)
can uniquely induce
de novo bone
formation
in a pro-fibrotic
microenvironment,
such as critical-size
bone defects
Guo, J., et al, Cytokine Growth Factor
Rev. 2012

20. Thanks to the rapid
progress of bone tissue
engineering techniques,
a large variety of novel
BMPs-based
bone-filling materials
have been developed to
significantly accelerate
and promote new bone
regeneration.
Guo, J., et al, Cytokine Growth Factor Rev. 2012

21. BMPs, a group of
proteinaceous growth
factors, were
discovered in the
pioneering work by
Urist in 1965
BMP family consists
of more than 30
members among which
19 BMPs are found in
human.
Urist, M.R. et al, Science 1965
Ducy, P. et al, Kidney Int. 2000

22. The classical role
for BMPs was
the induction of de
novo cartilage
and bone formation
in non-osseous sites
Urist, M.R., et al, Science 1965
Wang, E.A., et al, Proc. Natl. Acad. Sci. USA 1988

23. BMPs are currently
recognized as a group
of metabologens that
can orchestrate tissue
architecture
throughout the body
Reddi, A.H., et al, Cytokine Growth Factor Rev. 2009

24. BMPs can
significantly promote
the differentiation of
multipotent
mesenchymal stem
cells (MSCs) along
different lineages:
osteogenesis
adipogenesis
chondrogenesis
Levi, B., et al, Stem Cells 2011
Tseng, Y.H, et al, Nature 2008
Kim, H.J., et al, Tissue Eng. A 2009

25. The final
differentiation is
symbolized by
mineralization of
extracellular matrix
Zheng, Y., et al, Tissue Eng. A 2010
• Schematic graphs depicting the signaling pathways of bone
morphogenetic proteins (BMPs) and its-induced osteogenic
activities.
• BMPR-I: BMP type I receptors; BMPR-II: BMP type II
receptors; Runx2: runt-related transcription factor 2;
• ALP: alkaline phosphatase; OCN: osteocalcin; Dlx5: distal-
less homeobox 5;
• JNK: c-Jun N-terminal kinase; : clarified mechanisms; :
unclarified mechanisms.

26. Clinical Applications of BMPs

27. In USA, Europe and
Australia, BMP2 and
BMP7 have already
been approved for a
clinical application in
nonunion bone
fractures and spinal
fusions.
Bessa, P.C., et al, Tissue Eng. Regen. Med. 2008

28. In current clinic practice,
BMP2 is typically used
through its superficial
adsorption onto collagen
sponges
(e.g., INFUSE®).
Deproteinized bovine
bone was also rendered
osteoinductive using the
same.
Schwarz, F, et al, Clin. Oral Implants Res. 2008

29. However, this out-of-date
carrying mode of BMP2,
that was developed
decades ago, shows
a series of potential side
effects, such as
an over-stimulated
osteoclastic bone
resorption
and an excessive
ossification at unintended
sites
Shields, L.B. et al, Spine 2006
Toth, J.M, et al, Spine 2009

30. Co-Delivery Systems
for
Antibacterial
and
Osteoinductive Drugs
to
Repair Infected Bone Defects

31. This co-delivery system
should first fulfil the
general requirements for a
proper bone substitute:
good biocompatibility
sufficient mechanical
strength
high osteoconductivity
proper degradability

32. The system must
also be capable of
locally and slowly
delivering both
antibacterial
osteoinductive drugs

33. The drugs can be
combined with
biomaterials either by:
superficial adsorption
binding with a chemical
bond
by an internal
encapsulation
by a coating layer

34. For a co-delivery
system, both
antibacterial and
osteoinductive drugs
can be released either:
in a simultaneous mode
in a sequential mode

35. Schematic graph depicting
the four different carrying
modes of both antibacterial
and osteoinductive drugs in
biomaterials aiming to treat
infected bone defects.
(A) Superficial adsorption with
or without physicochemical
bonds;
(B) A co-encapsulation;
(C) A mixed carrying mode with
encapsulated antibacterial drugs
and superficially adsorbed
BMPs;
(D) surface coatings:
(D1) both drugs are immobilized by
chemical bonds;
(D2) both drugs are encapsulated in
coating layers with separation layers
for a controlled release.

36. Superficial adsorption
onto clinically used
materials is the most
common way to
apply bioactive
agents
(A) Superficial adsorption with or
without physicochemical bonds;

37. For example,
although BMPs
were found in
1965, BMPs can
be applied in clinic
only through their
adsorption onto
collagen
membrane.

38. Consequently, an
unphysiologically
high amount
(e.g., milligrams)
of BMPs has to be
applied to elicit the
osteoinductive effects
Park, D.K., et al, Spine 2013

39. However, the transiently
high amount is
associated with a series
of potential possible
side effects, such as an
over-stimulation
of local bone resorption
Toth, J.M., et al, Spine 2009

40. The introduction of
new materials that
bear physicochemical
binding sites
for drugs may render
the release of
adsorbed drugs much
slower.

41. A silica calcium
phosphate
nanocomposite as a
co-delivery system
for vancomycin and
BMP2, was used.
Pacheco, H., et al, J. Biomed. Mater. Res. A 2014

42. It was shown that
interactions between
PO4
-3 and negatively
charged moieties of
vancomycin
as well as between the
Si–O–Si functional
groups and BMP2
do happen
Pacheco, H., et al, J. Biomed. Mater. Res. A 2014
Si – O – Si

43. Co-Encapsulation
for a
Simultaneous Release

44. The release kinetics of
encapsulated drugs is mostly
simultaneous and is largely
dependent on
the permeability
the degradability of the
carrying materials.

45. A collagen was used as
a co-delivery system for
AgNPs and BMP2
Sun,C.Y, et al, Biotechnol. Lett. 2015
Kong, Y., et al, Nanoscale 2013
AgNPs

46. The
BMP2/AgNP/collagen
scaffold composites
showed a strong
antibacterial activity
without adversely
affecting
the adherence
or proliferation of bone
marrow-derived MSCs
(BMSCs). Bone marrow-derived MSCs (BMSCs).

47. Calcium sulfate, a quick
self-setting material, is
widely used as a bone-
defect-filling material.

48. It is also frequently
adopted as an
antibiotic carrier for the
treatment of infected
bone defects
It has many advantages
such as
low price full
biodegradability,
good biocompatibility and
high osteoconductivity
Kanellakopoulou, K., et al, Int. J. Antimicrob. Agents 2009
Sanicola, S.M., et al, J. Foot Ankle Surg. 2005

49. Calcium was used
sulfate to carry BMP2
and vancomycin
through an internal
co-encapsulation
Wang, Y., et al, Arch. Orthop. Trauma Surg. 2011

50. However, this kind of
materials usually forms a
solid block and lacks of
porous structure, which may
hinder the ingrowth of bone
tissues
BMP2 was first adsorbed
onto chitosan/calcium
phosphate microspheres,
which was thereafter
embedded into calcium
sulfate
Doty, H.A., et al, J. Mater. Sci. Mater. Med. 2014

51. Polymers are another
group of materials
that were introduced
biodegradable
polyurethane (PUR)
scaffolds to deliver
BMP2 and
vancomycin
Guelcher,S.A., et al, J. Orthop. Trauma 2011

52. The vancomycin release
kinetics consisted of two
phases:
The first burst release for a
week to protect the graft
from contamination
A subsequent sustained
release with over the
minimum inhibitory
concentrations for
Staphylococcus aureus for
2 months

53. A Mixed Carrying Mode
for a
Sequential Release

54. Antibacterial and
osteoinductive drugs
can also be delivered
by a carrying material
through different
carrying modes

55. For example, an
antibacterial drug is
encapsulated into a
carrying material with
an osteoinductive
drug superficially
adsorbed onto its
surface, or vice versa
AgNP’S

56. The two carrying modes can
realize different aims:
the former mode is mainly
aimed for promoting bone
regeneration with a
prevention of potential
infection,
while the latter mode is
mainly aimed for suppressing
an existing bacterial activity
and thereafter promoting
bone regeneration

57. Zein was used as a
major starch storage
protein found in corn,
as a carrying material
for
antibacterial HACC
(hydroxypropyl-
trimethyl ammonium
chloride chitosan)
(a quanternized
chitosan)
BMP2
Strobel, C., et al, J. Control. Release 2011
Zein

58. 10 wt % HACC was
encapsulated into
zein, which showed a
strong antibacterial
effect without
significantly
compromising cell
proliferation
HACC
Chitosan

59. The release of the
superficially adsorbed
BMP2 was
significantly slowed
down with the higher
ratio of mesoporous
silica SBA-15
nanoparticles
In addition, the
mesoporous silica
SBA-15 nanoparticles
also enhanced the cell
viability of human
MSCs
SBA-15

60. It was concluded that
Silica/HACC/zein
scaffolds with both
antibacterial and
osteoinductive
activities had an
immense potential
in orthopedics
and other biomedical
applications
Zhou, P., et al, Biomaterials 2014
Silica/HACC/zein scaffolds

61. Surface Coatings

62. Titanium metallic
implants are
biologically inert
cannot induce new
bone regeneration
inhibit bacterial
activity
Vlacic-Zischke,J., et al, Biomaterials 2011

63. Such a limitation makes
their implantation very
challenging when they are
used in the sites
with compromised bone
regeneration capacity
high infection risk

64. In these cases, it is in
great need to develop
implants with both
antibacterial
osteoinductive
functions
One approach is to
coat the surfaces of
implants
Ionic debris is additive to the
influences
of particulate debris and can have a
significant impact on local
cytotoxicity.

65. Attempts have been
done to prepare
either polymeric
coatings with
incorporated
antibacterial drugs
inorganic coatings
with incorporated
BMP2
Radin, S., et al, Biomaterials 2007
Wu,G., et al, TissueEng. CMethods2010
Wu, G., et al, Biomaterials 2010

66. By carefully combining
the principles of
polymeric
inorganic coatings
a co-delivery system was
developed for
antibacterial and
osteoinductive drugs,
which contained
electrochemically
deposited
chitosan/Ag/Haand
adsorbed haperin /BMP2
Xie, C.M., et al, ACS Appl. Mater. Interfaces 2014

67. This coating could
simultaneously
release Ag+ and
BMP2

68. A co-delivery system
with a sequential release
was introduced it was
manipulated the
concentrations and
sequences of
one polymer-[poly(D,L-
lactide)] (PDLLA), as a
sequential drug delivery
coating
with three distinctly
different release profiles
Cont.

69. A burst release of
gentamicin;
A burst release of IGF-
I followed by a
sustained release and
A slow release of
BMP2
Strobel, C., et al, J. Control. Release 2011

70. Gentamicin,
incorporated in the
outer layer, exhibited
a burst release profile,
which
was due to the very
thin 0.5 x or 1 x
PDLLA layers
and its direct expose to
an aqueous
environment

71. IGF-I (insulin-like
growth factor I),
in the middle layer,
exhibited
a fast release
and a subsequent slow
release, which was
controlled by the
erosion of the thicker
middle layer

72. BMP2, in the inner
layer, exhibited no
significant burst
release but as low and
sustained release
by this sandwich
approach
Strobel, C., et al, J. Control. Release 2011

73. Such a sequential release
profile was supposed to
exert the functions of
these drugs sequentially:
the rapidly-released
gentamicin to suppress
bacterial activities;
the secondly-released
IGF-1 to stimulate the
proliferation of
osteoblasts;
the slowly-released
BMP2 to enhance an
osteogenic differentiation.
Strobel, C., et al, J. Control. Release 2011

74. Using
a layer-by-layer
(LBL) principle, a
novel system was
introduced that could
realize a tunable
staged release of dual
drugs for orthopedic
implants
Min, J., et al, Biomaterials 2014

75. This multilayered coating
consists of two parts:
a base osteoinductive component
by dipping into a sodium acetate
solutioncontainingBMP2,poly(β-
aminoesters)(Mn~10kDa)
and poly(acrylicacid)(MW ~ 450
kDa);
an overlying antibacterial layer
by dipping into
a sodium acetate solution
containing gentamicin, poly(β-
amino esters) (Mn ~ 11 kDa)
and poly(acrylic acid) (MW ~ 1.25
MDa)
Min, J., et al, Biomaterials 2014

76. Cationic chitosan
or poly
(diallyldimethylammoni
um chloride) (MW ~
200–300 kDa)
and
anionic laponite clay
were alternately sprayed
onto both
osteoinductive
and
antibacterial layers
Min, J., et al, Biomaterials 2014
Cationic chitosan

77. Conclusions

78. The repair of infected bone
defects remains a formidable
challenge in the fields of:
oral implantology,
maxillofacial surgery
and orthopedics
Due to the less optimal efficacy
of current clinical treatments,
novel biomaterials with
both antibacterial
osteoinductive properties have been
developed in order to provide a
viable treatment option

79. In comparison with the clinically
used antibiotics, many
novel antibacterial biomaterials
showed very promising application
potential due to their
broader bactericidal spectrum,
nearly no resistance
and good biocompatibility
BMPs, particularly BMP2, are the
most potent osteoinductive drugs
to induce an in vitro
osteoblastogenesis
and an in vivo osteogenesis
Silver

80. The antibacterial and
osteoinductive drugs can be
incorporated into
co-delivery system through
the following modes:
Superficial adsorption /
binding with a chemical bond
An internal encapsulation
A mixed carrying mode with a
superficial adsorption and an
internal encapsulation
A surface coating

81. By manipulating the carrying
modes, the antibacterial and
osteoinductive drugs can be
released in varied modes
with different kinetics (burst or
slow)
and temporal characteristics
(simultaneous or sequential).
These novel biomaterials with
both antibacterial and
osteoinductive properties
showed very a promising
potential for clinical
applications.
BMPs
Silver nanoparticles