U.S. patent application number 17/374700 was filed with the patent office on 2021-11-04 for high strength intraosseous implants.
The applicant listed for this patent is UNIVERSITY OF CENTRAL OKLAHOMA. Invention is credited to Niyaf Nidhal Kadhem ALKADHEM, Rami Mohanad Mahdi ALKHALEELI, Morshed KHANDAKER, Shahram RIAHINEZHAD, Vagan TAPALTSYAN.
Application Number | 20210338383 17/374700 |
Document ID | / |
Family ID | 1000005712339 |
Filed Date | 2021-11-04 |
United States Patent
Application |
20210338383 |
Kind Code |
A1 |
TAPALTSYAN; Vagan ; et
al. |
November 4, 2021 |
HIGH STRENGTH INTRAOSSEOUS IMPLANTS
Abstract
The present invention enables modification of an intraosseous
implant device that is not only biologically non-inert, but can
stimulate bone and vascular growth; decrease localized
inflammation; and fight local infections. The method of the present
invention provides a fiber with any of the following modifications:
(1) Nanofiber with PDGF, (2) Nanofiber with PDGF+BMP2, and (3)
Nanofiber with BMP2 and Ag. Nanofiber can be modified with other
growth factors that have been shown to improve bone growth and
maturation--BMP and PDGF being the most common. Nanofiber can be
applied on the surface of the implant in several ways. First, a
spiral micro-notching can be applied on the implant in the same
direction as the threads with the nanofibers embedded into the
notches. Second, the entire surface of the implant may be coated
with a mesh of nanofibers. Third, it can be a combination of both
embedding and notching.
Inventors: |
TAPALTSYAN; Vagan; (El
Cerrito, CA) ; KHANDAKER; Morshed; (Edmond, OK)
; RIAHINEZHAD; Shahram; (Fort Lee, NJ) ;
ALKHALEELI; Rami Mohanad Mahdi; (Edmond, OK) ;
ALKADHEM; Niyaf Nidhal Kadhem; (Edmond, OK) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
UNIVERSITY OF CENTRAL OKLAHOMA |
Edmond |
OK |
US |
|
|
Family ID: |
1000005712339 |
Appl. No.: |
17/374700 |
Filed: |
July 13, 2021 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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16286005 |
Feb 26, 2019 |
11058521 |
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17374700 |
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16248122 |
Jan 15, 2019 |
10932910 |
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16286005 |
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15791571 |
Oct 24, 2017 |
10206780 |
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16248122 |
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15467652 |
Mar 23, 2017 |
9809906 |
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15791571 |
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14734147 |
Jun 9, 2015 |
10415156 |
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15467652 |
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15976615 |
May 10, 2018 |
10286103 |
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16248122 |
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15674309 |
Aug 10, 2017 |
9974883 |
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15976615 |
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62634993 |
Feb 26, 2018 |
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62312041 |
Mar 23, 2016 |
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62038506 |
Aug 18, 2014 |
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62373786 |
Aug 11, 2016 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61C 8/0013 20130101;
A61K 6/898 20200101; A61C 13/0018 20130101; A61C 8/0006 20130101;
A61C 8/0016 20130101; A61K 6/891 20200101; A61C 8/0022
20130101 |
International
Class: |
A61C 8/02 20060101
A61C008/02; A61C 8/00 20060101 A61C008/00; A61C 13/00 20060101
A61C013/00; A61K 6/891 20060101 A61K006/891; A61K 6/898 20060101
A61K006/898 |
Goverment Interests
STATEMENT AS TO RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSORED
RESEARCH OR DEVELOPMENT
[0002] This invention was made with government support under Grant
Number 5P20GM103447 awarded by the National Institutes of Health.
The government has certain rights in the invention.
Claims
1-17. (canceled)
18. An intraosseous dental implant, comprising: a threaded
endosseous dental device with a cylindrical shape and having an
external thread extending from a root of the device along a helix
angle with interspaces located between turns of the thread;
laser-grooved surfaces consisting of two parallel microgrooves at
the interspaces between the thread turns and oriented according to
the helix angle of said thread to provide a helix angle of the
microgroves, wherein said laser-grooved surfaces are coated with
tresyl chloride coupled with fibronectin (FN), wherein said
laser-grooved surfaces are further coated with a nanofiber matrix
(NFM), and wherein, said NFM is adhered to said laser-grooved
surfaces of said dental device, and aligned with the helix angle of
said microgrooves
19. The implant according to claim 18, wherein said microgrooves
exhibit a capacity to hold 18 layers of nanofibers where
microgroove depth is 5 .mu.m.
20. The implant according to claim 19, wherein said NFM comprises
18 fiber layers where said fibers are deposited circumferentially
along the helix angle of said microgrooves and shielded from
applied loads.
21. The implant of claim 18, wherein said microgrooves are engraved
to a depth of 5 .mu.m, a width of 50 .mu.m, and a spacing of 50
.mu.m to 150 .mu.m between said grooves at the interspace of said
thread.
22. The implant of claim 18, further comprising attached collagen
loaded with silver nanoparticles (Ag NP) or antimicrobial analogs
thereof.
23. The implant of claim 18, wherein said nanofiber matrix (NFM)
comprises at least one of growth factor or antibiotic-modified
polycapronlectron (PCL) Electrospun Nanofibers (ENFs), said PCL-ENF
combined with at least any of Platelet Derived Growth Factor
(PDGF), Bone Morphogenetic Protein 2 (BMP2), PDGF+BMP2, or BMP2 and
silver (Ag).
Description
CROSS REFERENCES TO RELATED APPLICATIONS
[0001] This application is a continuation of U.S. patent
application Ser. No. 16/286,005 filed Feb. 26, 2019 by the
University of Central Oklahoma (Applicant), entitled "Method and
apparatus for improving osseointegration, functional load, and
overall strength of intraosseous implants" which application is a
continuation-in-part and claims benefit of U.S. patent application
Ser. No. 16/248,122 filed Jan. 15, 2019 by the University of
Central Oklahoma (Applicant), entitled "Nanofiber coating to
improve biological and mechanical performance of joint prosthesis"
the entire disclosure of which is incorporated herein by reference
in its entirety for all purposes. This application claims the
benefit of U.S. Provisional Patent Application No. 62/634,993 filed
on Feb. 26, 2018 in the name of Vagan Tapaltsyan and Morshed
Khandaker, which is expressly incorporated herein by reference in
its entirety.
FIELD OF THE INVENTION
[0003] The present invention generally relates to the field of
polymer fiber production and biomedical applications thereof. More
specifically, the invention relates to improving performance of
metallic dental implants by attachment of augmented electrospun
fibers exhibiting micron to nano size diameters.
BACKGROUND OF THE INVENTION
[0004] Polycaprolecton (PCL) Electrospun Nanofibers (ENF) have
numerous biomedical applications. Co-pending application Ser. No.
14/734,147 and U.S. Pat. No. 9,359,694 by the present Applicant
discloses a method and apparatus for controlled deposition of
branched electrospun fiber on biomedical implants and material, the
disclosures of which are incorporated herein by reference in the
entirety. Research has shown that micron to nano size fibers may be
fused with biomedical implants for improving the mechanical and
biological adhesion of titanium implants with the host tissue. Nano
size fibers have been found to be excellent carriers of drugs for
improving bone growth. If applied as a coating around the implant,
improved bone growth may reduce the implant loosening problem. U.S.
Pat. Nos. 10,206,780 and 9,809,906 by the present Applicant
disclose methods to achieve adhesion of functional nanofiber
coatings on a biomedical implant surface to increase the
osteoinductive properties, and thereby to improve osseointegration
of an implant, the disclosures of which are incorporated herein by
reference in the entirety. The method uses PCL ENF fiber applied as
a coating material, forming an extracellular matrix on an implant
to improve ENF fiber adhesion with an implant surface, enabling use
at physiological load bearing conditions. The method supports
attachment of ENF fibers to an implant surface for both regular and
irregular shape implants and enables drug delivery to promote bone
growth.
[0005] The loss of teeth is a significant public health issue,
increasing the risk of a wide range of conditions such as
malnutrition due to lack of proper masticatory function and
clinical depression due to the change in facial appearance. Tooth
loss is primarily caused by periodontal disease, dental caries, or
trauma. The prevalence of all risk factors of tooth loss increases
with age and is thus projected to increase with the growth of the
aging population across the world. In the United States, the
population over 65 years of age is projected to reach 83.7 million
by 2050. Though the success rate of dental implant surgery is high,
the failure of implants due to poor osseointegration has been
reported.
[0006] Threaded endosseous devices with a cylindrical or tapered
shape are the most widely used type of dental implant. Endosseous
dental implants are surgically inserted into the jawbone.
Osseointegration refers to bone grown right up to the implant
surface without interposed soft tissue layer. Alveolar bone
osseointegrates with the implant without development of a
periodontal ligament. In cases of decreased primary stability of
the implant in bone, micro-motions occur at the implant surface
that lead to osteoclast-driven resorption of bone around the
implant, contributing to further implant loosening and eventual
implant failure. Delayed bone healing leads to potential failure of
the dental implant. Along with physical pain and suffering, implant
loosening due to poor osseointegration and healing leads to
economic burdens.
[0007] Healing, surgical success, and complete osseointegration are
regarded as the most important characteristics of dental (and, to
large extent, orthopedic) intraosseous implants. Currently, efforts
to improve implant success and osseointegration rates focus on the
mechanical aspects of implants, such as the type of alloy, taper,
screw thread design, metal finish (acid and laser etching,
polishing), etc. All intraosseous devices of the above classes are
classified as biologically inert implants, as implant integration
occurs through a process of bone remodeling, resulting in total
ankylosis. Another approach to improve osseointegration is the
direct attachment of osteoinductive nanoscale topographies on
endosseous dental implant surfaces. The main concern related to
coating nanoscale materials onto an implant surface is the risk of
coating detachment and toxicity of related debris. Further, implant
length and diameter are important in determining the stability of
the implant and the maximum load that can be placed on the implant.
Generally, at least 7-9 mm of bone depth is required for implant
placement, with implant width varying from 3-7 mm in diameter.
These dimensions often act as limiting factors when insufficient
bone is present for implant placement. Thus, there is a need for
stronger implants with higher functional loads and
osseointegration.
SUMMARY OF THE INVENTION
[0008] The present invention is directed to increasing success rate
of implant surgeries and decreasing integration time independent of
the physical properties of implants. The present invention is
unique in that it provides a method of incorporating a system of
bioaugmentation of metal implants by introducing a PCL ENF carrying
a recombinant growth factor, such as BMP2 (Bone Morphogenetic
Protein 2) or PDGF (Platelet Derived Growth Factor). These growth
factors have been extensively shown to activate neural
crest-derived bone stem cells to aid in differentiation,
regeneration, and maturation of bone. Moreover, the properties of
the fiber allow for binding to an antibiotic agent, such as silver
nanoparticles (Ag NP), thus making the implant itself to exhibit
antiomicrobial properties, decreasing the risk of implant failure
due to infection.
[0009] The aforementioned innovations can result in higher
stability and retention of intraosseous implants. Thus, while
current dental bio-inert implants require 7-9 mm of bone depth,
addition of modified PCL ENFs may decrease that requirement due to
growth factor-driven improved bone quality and improved
osseointegration. Also, current bio-inert implants require 4-6
months for complete osseointegration, while PCL ENF modification
may significantly reduce the osseointegration time and speed up
recovery. Finally, the present invention may allow for a wider
range of acceptable surgical sites for implant placement due to the
ENF's ability to stimulate new bone growth.
[0010] The present invention enables modification of an
intraosseous implant device that is not only biologically
non-inert, but can (1) stimulate bone and vascular growth (2)
decrease localized inflammation, and (3) fight local infections.
The method of the present invention provides a fiber with at least
any of the following modifications:
[0011] 1. Nanofiber with PDGF
[0012] 2. Nanofiber with PDGF+BMP2
[0013] 3. Nanofiber with BMP2 and Ag
[0014] Further, nanofiber can be modified with many growth factors
that have been shown to play a role in regulation of physiological
bone remodeling to improve bone growth and maturation. These may
include, for example, IGFs, TGF-.beta., FGFs, EGF, and WNTs, as
well as BMP and PDGF. Bone development, remodeling, and repair
requires attraction of mesenchymal progenitor cells (MPC) and
differentiation of MPC into osteoblasts. The effect of rhBMP-2,
rxBMP-4, and rhPDGF-bb as chemoattractive proteins for primary
human MPC has been shown to be highly significant. Thus, BMP and
PDGF are the most commonly ones employed.
[0015] Titanium-based implants have been widely used in orthopedics
and orthodontic surgeries because of their strong mechanical,
chemical and biological properties. We have tested a set of steps
(e.g. grooving and oxidizing) by which a nanofiber matrix (NFM),
composed of collagen (CG) and poly-.epsilon.-caprolactone (PCL)
electrospun nanofibers, can be coated on a Ti implant without
subsequent detachment. A significantly improved osseointegration of
CG-PCL NFM-coated Ti over non-coated Ti not previously known was
observed in our experiments. The advantage of functional coating
treatment on an implant is that it is simple, indirect, scalable,
inexpensive, and supplementary to other surface treatment
techniques. Such treatment can be applied on an implant surface
without affecting other implant factors, such as mechanical,
medication (e.g. drugs, irradiation), and patient (e.g. age,
osteopenia) factors. The biological properties of a functional
coating can be further improved by adding growth factors, proteins,
and other molecules to create a truly osteoinductive platform at
the implant/bone interface.
[0016] In one major aspect the present invention provides an
improved intraosseous implant device capable of decreasing the time
periods for osseointegration and preventing post-operative local
infections. Titanium (Ti) alloy is most widely used as a dental
implant material. We have developed a novel method of coating
cylindrical Ti implants with nanofiber mesh by microgrooving. We
have also immobilized fibronectin (FN), a glycoprotein of the
extracellular matrix, on a Ti alloy (Ti-6Al-4V) by tresyl
chloride-activation method. Our studies show that microgrooving on
cylindrical Ti implants and subsequent coating of the grooves with
collagen-poly-.epsilon.-caprolactone nanofiber matrix (CG-PCL NFM)
significantly improves the biocompatibility, mechanical stability
and osseointegration of Ti. A laser pulse can create microgrooves
on the surface of regular-and irregular-shaped implants. A
literature search has revealed no reported research directed to the
controlled fabrication of microgrooves on a complex-shaped implant
surface, such as a dental implant (FIG. 1). The effect of coating
the laser-induced microgrooves with bone morphogenetic protein-2
(BMP2)- and silver (Ag) nanoparticle (NP)-immobilized PCL NFM on a
dental implant has not been reported. Attachment of BMP2 and Ag NP
onto Ti dental implant is sought via FN and CG immobilized PCL NFM,
respectively. The above mentioned surface treatments on a dental
implant may have the potential to improve the implant
osseointegration, reducing both healing time, and risk of
infection.
[0017] In another major aspect the present invention provides
methods for attachment of osseointegration-promoting and
anti-bacterial biomolecules on a dental implant using laser-induced
microgrooves and PCL NFM. Immobilization of BMP2 with PCL NFM
(referred as BMP2-PCL) and subsequent coating of a
laser-microgrooved titanium implant by BMP2-PCL may lead to greater
in vitro and in vivo osteogenic functions in comparison to the
non-treated implants due to higher biological compatibility of the
BMP2-PCL-coated implant.
[0018] Immobilization of Ag NP with PCL NFM (referred as Ag-PCL)
and subsequent coating of a laser-microgrooved titanium implant by
Ag-PCL may lead to lower risk of bacterial development in
comparison to the non-treated implants due to higher anti-bacterial
resistance of the Ag-PCL-coated implant.
[0019] In another major aspect the present invention provides
methods for fabrication of the control microgrooves at the
interspace between two threads of a dental implant. Fabrication of
such grooves can have medical benefits, since grooves on implants
induce a higher amount of implant--bone contact area and osteoblast
cell function in comparison to implants without grooves. During
implantation, the implant body is strongly torqued and drilled into
hard bone. The microgrooves protect the functional NFM coating from
these applied loads. The NFM coating can serve as a reservoir at
the microgrooves on dental implant surfaces for controlled release
of bone growth factor and anti-bacterial molecules for reducing
infection and promoting osteogenesis. Laser pulse is a method used
to produce high precision, high roughness, and uniform microgroove
topography on a dental implant along the threaded and non-threaded
sections.
[0020] In another major aspect the present invention provides
methods for attachment of the functional PCL NFM coating on a
dental implant of any size or shape. Tresyl chloride, a chemical
activation technique, can be used to attach FN on the dental
implant surface directly. The bone growth signaling factors and
collagen binding domain of FN can be utilized to attach bone growth
factors (BMP2) and anti-bacterial molecules (Ag NP) immobilized PCL
NFM with Ti. The combined effect of FN immobilization on
laser-microgrooved Ti and coating those grooves by PCL NFM provides
a novel technique to attach the functional PCL NFM coating on any
Ti implant.
[0021] In another major aspect the present invention provides
methods for a direct immobilization technique using bone growth
factors (BMP2) and anti-bacterial molecules (Ag NP) on a human
dental implant surface via PCL NFM. A potential opportunity for the
advancement of in-vivo tissue-to-implant osseointegration, faster
healing times, and reduction of infection of a dental implant are
possible from these inventions. Such treatment methods can be
applied not only to improve dental implant-to-bone interface, but
also to anchor many other orthopedic biomaterials.
[0022] Nanofiber can be applied on the surface of an implant in
several ways. First, a spiral micro-notching can be applied on the
implant in the same direction as the threads, with the nanofibers
embedded into the notches. Second, the entire surface of the
implant may be coated with a mesh of nanofibers. Third, it can be a
combination of both embedding and notching.
BRIEF DESCRIPTION OF THE DRAWINGS
[0023] FIG. 1 is a non-limiting diagram showing microgrooves and
nanofiber-assisted drug delivery on dental implant as provided by
the present invention.
[0024] FIG. 2 is a non-limiting diagram showing the method of the
present invention providing protein immobilization on Ti using
nanofiber matrix as a functional coating.
[0025] FIG. 3 is a non-limiting diagram showing F1s, S2p, N1s and
O1s spectra of the Ti, Tresyl/Ti, and FN/Ti surface by a XPS
analysis.
[0026] FIG. 4 is a non-limiting diagram showing precision
microgrooves on the flat surface of a Ti rod fromed by the method
of the present invention.
[0027] FIG. 5 is a non-limiting diagram showing schematic
representation of the processes for preparing an in vivo dental
implant.
[0028] FIG. 6a is a non-limiting diagram showing a 3 mm diameter
screw coated with 18 layers of PCL NFM.
[0029] FIG. 6b is a non-limiting diagram showing twisting of an NFM
coated screw in to a pre-drilled hole (2.6 mm diameter) on a clear
acrylic shows homogenous distribution of fiber along screw/acrylic
interface.
[0030] FIG. 7a is a non-limiting image showing individual
immobilization of CG and FN on PCL NFM.
[0031] FIG. 7b is a non-limiting graph showing that the individual
immobilization of CG and FN on PCL NFM has no adverse effect on
osteoblast cells adhesion and proliferation of PCL NFM, and
significant increase of cell adhesion observed for FN-PCL-NFM when
compared to PCL NFM (p<0.05).
[0032] FIG. 8a is a non-limiting graph showing a gradual increase
of release of BMP2 for 28 days was observed for FN-Hep-BMP2/PCL
samples.
[0033] FIG. 8b is a non-limiting image showing cell divisions after
48 hours of cell culture on FN-BMP2-PCL samples.
[0034] FIG. 9a is a non-limiting image showing PCL samples after
Gram staining.
[0035] FIG. 9b is a non-limiting image showing CG-Ag-PCL samples
after Gram staining.
[0036] FIG. 10 is a non-limiting diagram presenting the test
results of time-dependent bone growth around the CG-PCL NFM-coated
Ti implant and related in vivo pull-out tests to demonstrate
mechanical stability of microgrooved --Ti.
DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS OF THE INVENTION
[0037] Our research demonstrates: (1) immobilization of ECM
proteins (CG and FN) and bone growth factors (BMP2) with PCL NFM is
possible, and such immobilization improves the in vitro cell
viability of PCL NFM; (2) immobilization of antibacterial
nanoparticles (Ag) with PCL NFM is possible, and such
immobilization improves the in vitro antibacterial activity of PCL
NFM; (3) direct attachment of FN on a dental implant material
(Ti-6Al-4V) is possible using tresyl chloride activation method;
and (4) microgrooving of a Ti implant followed by coating the
microgrooves with CG-PCL NFM significantly improves in vivo
mechanical stability and osseointegration.
[0038] Referring to FIG. 1, in a preferred embodiment the present
invention 10 provides coating methods described above to dental
implants with the goal of improving the osseointegration of
implants. We use a laser pulse to create microgrooves 11 at the
interspace between two threads 18 of a dental implant (Di) 12.
Laser-induced microgrooves 11 were shown in our research to
significantly influence the surface morphology, contact angle,
surface roughness, and chemical composition of Ti that can
influence the attachment of fibronectin on implants. A set of
continuous microgrooves 11 with 50 .mu.m width, 5 .mu.m depth, and
150 .mu.m spacing between grooves are engraved at the root of the
threads (.about.0.5 mm) on a Di 12 using a laser system (e.g., a
Galvo FP fiber marking) to produce a laser-microgrooved Di 13. A
rotary stage of the laser system (not shown) is oriented according
to the helix angle of the implant threads to produce the laser
microgrooves 11 at the root 14 of the threads 18.
[0039] Referring to FIG. 2, in a preferred embodiment FN is attach
on the laser-engraved implant 12 surface by tresyl chloride method
21, and coats the FN-immobilized implant surface 22 with BMP2- and
Ag-immobilized PCL NFM as shown 23. Basic terminal hydroxyl groups
of a pure titanium surface 22 react with tresyl chloride, which
allows for further coupling with fibronectin (FN). Previous in vivo
studies using a rabbit femur model found that immobilizing
fibronectin (FN) onto cylindrical pure titanium implants enhanced
bone regeneration around implants. However, pure titanium has
limited applications in the biomedical industry due to its inferior
mechanical and biological properties, compared to biomedical grade
titanium alloys, such as Ti-6Al-4V (the most commonly used titanium
alloy in medical devices). We examined whether human plasma FN can
be attached to Ti-6Al-4V via the tresyl chloride activation method.
Three groups of samples were prepared to test the FN attachment on
Ti via the tresyl chloride activation process: (1) control, (2)
tresyl chloride-activated Ti (referred to as Tresyl/Ti), and (3)
tresyl chloride-activated Ti subsequently coupled with FN (referred
to as FN/Ti). To prepare Tresyl/Ti, the top surface of a polished
Ti-6Al-4V sample was treated with 2,2,2-Trifluoroethanesulfonyl
chloride at 36.degree. C. for 48 hours, then washed with water,
water-acetone (50:50), and acetone. Samples were then dried and
stored in a desiccator. To prepare FN/Ti, a Tresyl/Ti sample was
treated for 24 hours at 37.degree. C. with human plasma fibronectin
diluted in phosphate-buffered saline (PBS) solution to a
concentration of 0.1 mg/mL. X-ray photoelectron spectroscopy (XPS)
analysis was conducted on all samples to determine the chemical
state of Ti. The binding energy for each spectrum was calibrated
against the Cls peak at 284.8 eV.
[0040] Referring to FIG. 3, XPS analysis found the presence of an
amide group for FN/Ti, which confirms the surface activation by
tresyl chloride and then direct coupling of FN with Ti. The N1s
peak, derived from the amide bond of immobilized fibronectin, was
detected around the binding energy of 400 eV for only FN/Ti
samples. Therefore, this study suggested that direct attachment of
FN is possible on a tresylated Ti alloy surface. Our proof of
concept for the potential application of the treatment protocols on
a Di led to the following: an ideal functional coating for a Di
must reabsorb with time to allow and encourage new bone formation
while maintaining its osteoconductive properties in vivo.
[0041] Referring to FIG. 4, the present invention provides laser
engraving 41 to support attachment of FN on a dental implant (FIGS.
1-12) and then coating the implant with BMP2 and Ag NP-immobilized
PCL NFM. A laser pulse can be applied on the polished surfaces of
Ti to create linear and continuous microgrooves on Ti implants. A
laser capable of producing precision microgrooves (FIG. 1-11) on at
least the flat surface of a Ti rod 42 can be used for this purpose.
In our research we have used a Galvo FP fiber marking laser
equipped with software for engraving a set of microgrooves (10
.mu.m width, 5 .mu.m depth, and 50 .mu.m spacing between grooves)
on Ti. The reason for achieving 5 .mu.m-deep microgrooves on Ti is
due to the fact that each groove of this size can accommodate at
least 18 layers of nanofiber (average fiber diameter .about.300
nanometers). In the present invention, we use 18 layers of PCL NFM
because our research shows that the porosity of a PCL NFM membrane
comprising 18 layers of fibers is adequate for cells to migrate
through the membrane. FN can be attached on laser engraved Ti
implants (lgTi), where immobilization of FN with Ti is accomplished
by the tresyl chloride method as provided by the present
invention.
[0042] Referring to FIG. 5, aligned unidirectional PCL NFM can be
collected using the methods disclosed in U.S. Pat. No. 9,809,906 by
the present Applicant and illustrated in FIG. 5. The laser-grooved
surface of lgTi is activated by tresyl chloride and then 18 layers
of PCL NFM is deposited along the direction of the thread
(clockwise) by rotating the tresylated Di 18 times until the
implant collects 18 layers of fibers. The reason for adapting this
coating method on a Di surface is due to fact that such a method
should be able to maintain nanofibers along the Di/bone interface.
FN, FN-Hep-BMP2 and CG-Ag complexes are gently splashed on the PCL
coated Di samples to prepare FN-PCL/Di, FN-BMP-PCL/Di and
CG-Ag-PCL/Di Di, respectively. All implants are prepared under
sterile conditions and kept for 30 minutes in a portable
ultraviolet sterilizer before surgery.
[0043] Referring to FIGS. 6a and 6b, in a method validation test we
coated a M3.times.0.5 screw by PCL NFM using the method of the
present invention (FIG. 5). We torqued the fiber-coated screw in to
a pre-drilled hole (2.6 mm) on clear acrylic (FIG. 6b). We observed
homogeneously-distributed fiber along the interface between the
screw and the acrylic (FIG. 6b).
EXPERIMENTAL ASPECTS
Immobilization of Bone Morphogenic Protein-2 (BMP2) on Ti Using
Fibronectin and PCL NFM.
[0044] Bone morphogenic proteins (BMPs) play important roles in in
osteoblast and chondrocyte differentiation. Research shows that
surface functionalization of Ti with BMP2 improves the osteoblast
activities of Ti. Among BMP family members, BMP2 is a potent
osteoinductive factor that plays key role during bone formation.
Fibronectin (FN) is a multifunctional protein most abundantly found
in the extracellular matrix (ECM) under dynamic remodeling
conditions such as bone healing and development. Research shows
that tethering of FN onto Ti effectively enhanced the bone
regeneration around implants. Our preliminary studies show that
FN-immobilized PCL NFM (referred as FN-PCL) has higher
biocompatibility with osteoblast cells in comparison to PCL. FN
contains binding domains for many bone growth signaling factors,
including BMP2 and transforming growth factor-beta (TGF-.beta.). We
have successfully immobilized BMP2 with PCL NFM using FN in our
preliminary studies. The effect of BMP2-immobilized PCL NFM coating
on the osteogenic functions of Ti is not known and thus it needs to
be investigated.
Immobilization of Silver Nanoparticles (Ag NP) on Ti Using Collagen
and PCL NFM.
[0045] Prolonged anti-bacterial activities of an implant are
possible by tethering anti-bacterial molecules with the implant.
Many studies reported that Ag NP inhibits bacterial growth, while
retaining/promoting osteoblast viability. Among common
antibacterial nanoparticles (Ag, CuO, ZnO), Ag NP shows the minimum
toxicity to environmentally relevant test organisms and mammalian
cells in vitro and in vivo. Since Ag NP dissolves in CG, it can be
immobilized with CG-PCL NFM. Our in vivo and in vitro studies show
that CG-PCL NFM coating enhanced biological functions of Ti. This
is due to the fact that higher cell functions were created via
better cell signaling arising from the cell-cell contact and the
cell-NFM components in the case of the CG-PCL NFM-coated Ti samples
than non-coated Ti samples. Our preliminary studies showed no
antimicrobial activity of Ag NP-immobilized CG-PCL NFM towards
Staphylococcus aureus in comparison to PCL NFM. The effect of Ag
NP-tethered CG-PCL NFM on the osteogenic and anti-bacterial
activities towards other common aerobic bacterial organisms on Ti
implant is not known and thus needs to be investigated.
Effect of Immobilization of Fibronectin and Collagen on the
Cellular Functions of PCL NFM
[0046] Fibronectin (FN) contains several active sites, known as the
heparin-binding domains, collagen-binding domain, fibrin-binding
domain, and cell-binding domain, that serve as platforms for cell
anchorage. The goal of this preliminary study was to evaluate the
effect of immobilization of collagen and plasma fibronectin with
PCL NFM on the cellular functions of PCL NFM. The results (FIG. 7a
and FIG. 7b) show that the individual immobilization of CG and FN
on PCL NFM has no adverse effect on osteoblast cells adhesion and
proliferation of PCL NFM, although a significant increase of cell
adhesion was observed for FN-PCL-NFM when compared to PCL NFM
(p<0.05). A significant improvement of cell adhesion and
proliferation was observed for FN-CG-PCL NFM in comparison to PCL
NFM (p<0.01). This is due to the fact that higher cell functions
were created via better cell signaling arising from the cell-cell
contact and the cell-NFM components in the case of FN-CG
immobilized PCL NFM compared to PCL NFM.
[0047] Direct attachment of FN on a Ti implant surface is possible
using a Tresyl Chloride-Activated Method (shown in Section C.5.).
Since FN contains a CG binding domain, FN-immobilized Ti can
therefore be polymerized into CG-PCL. The effect of the attachment
of PCL NFM with Ti using CG and FN on the osteogenic functions of
the implant is not known and needs to be investigated.
Immobilization of Human Bone Morphogenic Protein-2 (BMP2) with PCL
NFM Using Fibronectin (FN).
[0048] The PCL NFM can be modified with heparin (Hep) and further
immobilized with BMP2. The modified fibers showed the potential to
effectively induce osteogenic differentiation of periodontal
ligament cells. Since FN contains heparin-binding domains, PCL
fibers can be modified with FN-Hep-BMP2 complex. The purpose of
this preliminary study was threefold: (1) to immobilize BMP2 on PCL
NFM using only FN-BMP2 and FN-Hep-BMP2 complexes, (2) to determine
the amount of BMP2 release from the immobilized BMP2-PCL NFM, and
(3) to compare the cell viability of BMP2-immobilized PCL NFMs with
respect to PCL NFM (control). Immobilized BMP2 was released from
the PCL NFMs in a sustained manner for 28 days, although the rates
of release of BMP2 from FN-BMP2/PCL and FN-Hep-BMP2/PCL were
different. A gradual increase of release of BMP2 for 28 days was
observed for FN-Hep-BMP2/PCL samples (FIG. 8a). Rapid release of
BMP2 for first 4 days, then gradual decline of release of BMP2 with
time was observed for FN-BMP2/PCL samples. Cells displayed a
well-extended morphology on all the BMP2-treated groups, when they
were compared with the control group (8a). FIG. 8b depicts a
representative image showing cell divisions after 48 hours of cell
culture on FN-BMP2-PCL samples. In the image, blue color shows the
attachment of cells on NFM. There was more than a 52% and 30%
increase in the cell viability on FN-Hep-BMP2-PCL samples after
culturing the cells for 72 hours compared to control and
FN-BMP2-PCL. Both release and cell viability tests suggested an
advantage of FN-Hep-BMP2 over FN-BMP2 complex for the
immobilization of BMP2 with PCL NFM. FN-Hep-BMP2 immobilized PCL
NFM has the potential to induce osteogenic differentiation of
osteoblast cells on a Ti implant surface, which is not yet known.
The effect of the treatment of Ti with FN by tresyl chloride method
and then coating by FN-Hep-BMP2/PCL on the osteogenic functions
needs to be investigated.
Attachment of Silver Nanoparticles (Ag NP) with PCL NFM Using
CG
[0049] Silver nanoparticles (Ag NP) show promising anti-bacterial
properties with biocompatibility and minimal toxicity. Ag NP-loaded
collagen was immobilized with polymeric film to inhibit bacterial
growth while promoting osteoblast cell viability. The
anti-bacterial activities of PCL NFM can be improved by
immobilizing Ag NP-loaded CG with PCL NFM. The purpose of this
preliminary study was to examine the effect of immobilization of Ag
NP-loaded CG on the anti-bacterial properties of PCL NFM. We
succeeded in immobilizing Ag-loaded collagen with PCL NFM. The SEM
and XRD analysis before and after 2 days of bacterial culture
confirmed the presence of Ag with PCL. Our bacterial culture
studies showed no sign of colonies growing on Ag-CG-PCL, whereas
the presence of bacteria was observed in PCL. FIG. 9 shows PCL
samples after Gram staining: (a) PCL and (b) CG-Ag-PCL. S. aureus
that take up the Gram stain were present in PCL, as observed in the
image by circular black shapes (pointed by arrows), while CG-Ag-PCL
without S. aureus, did not stain and appears with a gray color. One
reason for this might arise from an increased carrying capacity of
Ag NP-loaded collagen by the nanofiber disc due to its unique
surface-to-volume ratio.
In Vivo Evaluation of Coating a Titanium Implant with CG-PCL
NFM
[0050] We have invented a method of coating a cylindrical metal
implant with NFM that is made with CG-PCL (U.S. Pat. No.
9,809,906). Our invention implements a set of grooves that are
created on Ti in a circumferential direction to increase the
contact area between the implant and bone. CG-PCL NFM is
subsequently coated along the sub-micrometer grooves on the Ti
implant using our unique electrospinning process (U.S. Pat. No.
9,359,694). The goal of this research was to evaluate the effect of
CG-PCL NFM coating on the mechanical stability and osseointegration
of a Ti implant using a rabbit model. Our in vivo pull-out tests
demonstrated that mechanical stability of microgrooved --Ti was
significantly higher compared to non-grooved Ti. The mechanical
stability (quantified by shear strength) of groove-NFM Ti/bone
samples were significantly greater compared to other samples
(p<0.05). The pull-out strength of groove-NFM-coated Ti was
comparable to other functional coating-treated Ti reported in the
literature. The types of new bone growth on Ti was different
between groove and groove-NFM samples, which was observed from the
stained images of histology-sectioned images (FIG. 10e).
Histomorphometric results showed that the amount of BIC on Ti was
higher for groove-NFM (12.18.+-.0.94 mm, n=2) than groove
(5.30.+-.4.01 mm, n=2) samples. Due to the poor attachment of Ti
with bone, Ti implants came out from their implant sites during
histology preparation. Therefore, there was no result for any
control sample. Both .mu.CT analyses (FIG. 10e and FIG. 10f) and
blood serum (FIG. 10g) results confirmed the time-dependent bone
growth around the CG-PCL NFM-coated Ti implant and determined that
6 weeks were required for sufficient bone growth around the
implant.
[0051] Our in vivo pull-out tests demonstrated that mechanical
stability of microgrooved --Ti was significantly higher compared to
non-grooved Ti (FIG. 10c). The mechanical stability (quantified by
shear strength) of groove-NFM Ti/bone samples were significantly
greater compared to other samples (p<0.05). The pull-out
strength of groove-NFM-coated Ti was comparable to other functional
coating-treated Ti reported in the literature. The types of new
bone growth on Ti was different between groove and groove-NFM
samples, which was observed from the stained images of
histology-sectioned images (FIG. 10e). Histomorphometric results
showed that the amount of BIC on Ti was higher for groove-NFM
(12.18.+-.0.94 mm, n=2) than groove (5.30.+-.4.01 mm, n=2) samples.
Due to the poor attachment of Ti with bone, Ti implants came out
from their implant sites during histology preparation. Therefore,
there was no result for any control sample. Both .mu.CT analyses
(FIG. 10e and FIG. 10f) and blood serum (FIG. 10g) results
confirmed the time-dependent bone growth around the CG-PCL
NFM-coated Ti implant and determined that 6 weeks were required for
sufficient bone growth around the implant.
[0052] Further modifications and alternative embodiments of various
aspects of the invention will be apparent to those skilled in the
art in view of this description. Accordingly, this description is
to be construed as illustrative only and is for the purpose of
teaching those skilled in the art the general manner of carrying
out the invention. It is to be understood that the forms of the
invention shown and described herein are to be taken as examples of
embodiments. Elements and materials may be substituted for those
illustrated and described herein, parts and processes may be
reversed, and certain features of the invention may be utilized
independently, all as would be apparent to one skilled in the art
after having the benefit of this description of the invention.
Changes may be made in the elements described herein without
departing from the spirit and scope of the invention as described
in the following claims.
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