Porous Tantalum Coatings Prepared by Vacuum Plasma Spraying Enhance BMSCs Osteogenic Differentiation and Bone Regeneration In Vitro and In Vivo
et al. (2013) Porous Tantalum Coatings Prepared by Vacuum Plasma Spraying Enhance BMSCs Osteogenic
Differentiation and Bone Regeneration In Vitro and In Vivo. PLoS ONE 8(6): e66263. doi:10.1371/journal.pone.0066263
Porous Tantalum Coatings Prepared by Vacuum Plasma Spraying Enhance BMSCs Osteogenic Differentiation and Bone Regeneration In Vitro and In Vivo
Ze Tang 0
Youtao Xie 0
Fei Yang 0
Yan Huang 0
Chuandong Wang 0
Kerong Dai 0
Xuebin Zheng 0
Xiaoling Zhang 0
Jan Peter Tuckermann, Leibniz Institute of Age Research - Fritz Lipmann Institute, Germany
0 1 The Key Laboratory of Stem Cell Biology, Institute of Health Sciences, Shanghai Institutes for Biological Sciences (SIBS), Chinese Academy of Sciences (CAS) and Shanghai Jiao Tong University School of Medicine (SJTUSM) , Shanghai , P.R. China , 2 Shanghai Key Laboratory of Orthopaedic Implant, Department of Orthopaedic Surgery, Shanghai Ninth People's Hospital, Shanghai Jiao Tong University School of Medicine (SJTUSM) , Shanghai , P.R. China , 3 Shanghai Institute of Ceramics, Chinese Academy of Sciences , Shanghai , P.R. China
Tantalum, as a potential metallic implant biomaterial, is attracting more and more attention because of its excellent anticorrosion and biocompatibility. However, its significantly high elastic modulus and large mechanical incompatibility with bone tissue make it unsuitable for load-bearing implants. In this study, porous tantalum coatings were first successfully fabricated on titanium substrates by vacuum plasma spraying (VPS), which would exert the excellent biocompatibility of tantalum and alleviate the elastic modulus of tantalum for bone tissue. We evaluated cytocompatibility and osteogenesis activity of the porous tantalum coatings using human bone marrow stromal cells (hBMSCs) and its ability to repair rabbit femur bone defects. The morphology and actin cytoskeletons of hBMSCs were observed via electron microscopy and confocal, and the cell viability, proliferation and osteogenic differentiation potential of hBMSCs were examined quantitatively by PrestoBlue assay, Ki67 immunofluorescence assay, real-time PCR technology and ALP staining. For in vivo detection, the repaired femur were evaluated by histomorphology and double fluorescence labeling 3 months postoperation. Porous tantalum coating surfaces promoted hBMSCs adhesion, proliferation, osteogenesis activity and had better osseointegration and faster new bone formation rate than titanium coating control. Our observation suggested that the porous tantalum coatings had good biocompatibility and could enhance osseoinductivity in vitro and promote new bone formation in vivo. The porous tantalum coatings prepared by VPS is a promising strategy for bone regeneration.
Funding: This work was supported by grants from: The Ministry of Science and Technology of China (No.2011DFA30790, 2010CB945600), www.most.gov.cn;
National Natural Science Foundation of China (No. 81190133, 31101056), http://www.nsfc.gov.cn; Chinese Academy of Sciences (No.XDA01030404,
KSCX2-EW-Q1-07), http://www.cas.cn/; Science and Technology Commission of Shanghai Municipality (No.11QH1401600), www.stcsm.gov.cn; and Shanghai Municipal
Education Commission (No.J50206, 10SG22), www.shmec.gov.cn/. The funders had no role in study design, data collection and analysis, decision to publish, or
preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
In the last few years, great interest has been focused on tissue
engineering as a potential therapeutic approach for
musculoskeletal diseases. The role of metallic implants either for
osteosynthesis or for arthroplasty has been tested in preclinical and
clinical settings. An ideal implant material should have
appropriate elastic modulus, corrosion resistance, good biocompatibility
and favor bone anchorage. However, most medical implant
materials do not simultaneously fulfill all these characteristics.
Accordingly, various coatings have been developed to improve the
biocompatibility and osseoinductivity of load-bearing materials.
Hydroxyapatite (HA) coating is one of outstanding examples of
such coatings. HA-coated titanium (Ti) implants have the good
biological activity of HA and the excellent mechanical properties
of Ti alloy, and are thus widely used in orthopedic surgery as bone
replacement materials . Unfortunately, HA and other ceramic
coatings are brittle and have the problem of debond from
loadbearing substrate materials [2,3], which hinder their extensive
application. Thus, new type of coatings with good
biocompatibility, osseoinductivity, long-term chemical stability, and firm
bonding with substrate are still demanded for medical implants.
Tantalum (Ta) may be a potential coating material for medical
implants. Ta (atomic number 73) is a rare transition metal that is
highly corrosion resistant and inert in vivo . It has been used in
medical practice since the mid-1900s, and shows good medical
biocompatibility and safety [2,5]. Ta is considered as a potential
biomaterial given its excellent chemical stability, body fluid
resistance, biocompatibility, and biologic fixation with bone tissues
. However, its significantly high elastic modulus and large
mechanical incompatibility with bone tissue make it unsuitable as
bulk medical implant. Creating a porous layer on implant surfaces
may enable the medical applications of Ta. Trabecular MetalTM is
one such open cell porous Ta marketed by Zimmer Inc. (Warsaw,
IN, USA) [14,15]. These porous Ta components offer a low
modulus of elasticity, high surface frictional characteristics, and
Figure 1. Identification and lineage differentiation potential of hBMSCs. (A) hBMSC identification with the surface markers CD29+, CD342,
CD105+, CD44+, CD452, and CD90+. (B) The hBMSC lineage differentiation: osteoblasts (ALP stain), and adipocytes (Oil Red O stain).
excellent osseointegration properties (i.e., bioactivity,
biocompatibility, and in-growth properties) [6,13]. However, the relatively
high cost and difficulty in fabrication (i.e., processing in an inert
atmosphere, non-solderability, grinding difficulty, and high
melting temperature) of Ta restrict its widespread use in medical
practice . In this study, vacuum plasma spraying (VPS) was
first used to fabricate porous Ta coatings on titanium (Ti)
substrates. The VPS technology is an economical method for
fabricating porous coatings at very high melting temperatures. The
porous structure of Ta coatings can effectively alleviate the
mechanical incompatibility between Ta implants and host bone
tissues. Thus, porous Ta-coated Ti implants, which combine the
excellent biocompatibility of Ta and the good mechanical
properties of Ti, can be used in medical implants.
Bone repair requires a cellular source with the ability of
differentiation into bone together with a scaffold that allows the
adhesion and proferation of these cells. Bone marrow
mesenchymal stem cells (BMSCs) research is currently an exciting area of
interest, since these cells have the ability to differentiate into
several cell types, including osteoblasts, chondrocytes and
adipocytes. In this regard, they have been extensively assessed for bone
defects treatment . This study aimed to investigate the
biocompatibility, osteogenesis, and osseointegration of Ta coatings
applied by VPS. Human bone marrow stromal cells (hBMSCs)
were used for in vitro cellular adhesion, proliferation, and
Figure 2. XRD and surface morphology identification of Ta and Ti coatings. (A) XRD patterns (B) contact angle of porous Ta and Ti coatings
(t-test, assuming unequal variances, error bars represent 6 SD, n = 3; Ti coating, 131.91765.10882; Ta coating, 130.45565.55294; Prob.|t|, 0.7125
means no significant difference). (C) SEM images of the initial surface morphology of both coatings at 3006, 5006, 10006, and 30006
Figure 3. Morphology of hBMSCs cultured on Ta and Ti coatings. (A) SEM images of the surface morphology of both coatings with cultured
hBMSCs at 8006 magnifications. (B) Confocal laser scanning microscopy images of single hBMSC F-actin cytoskeletal morphology on both coatings
for 3 and 24 h. (red, phalloidin for F-actin; blue, DAPI for nucleus).
osteogenetic differentiation evaluation. Rabbit femur implant
models were used to evaluate the osseointegration and new bone
formation ability of porous Ta coating in vivo. This would be of
great impact to support its application for clinical purposes,
especially in the bone reconstructive techniques.
Materials and Methods
hBMSCs (Human bone marrow stromal cells) were donated by
patients with written informed consent and this experiment was
approved by Independent Ethics Committee of Shanghai Ninth
Peoples Hospital affiliated to Shanghai Jiao Tong University
School of Medicine (SJTUSM). The rabbits were obtained from
the Laboratory Animal Center of Shanghai Ninth Peoples
Hospital affiliated to Shanghai Jiao Tong University School of
Medicine (SJTUSM) (Certificate number SCXK 2007-0007).
Handling of the animals was in accordance with policies of
Shanghai Ninth Peoples Hospital affiliated to Shanghai Jiao Tong
University School of Medicine (SJTUSM) and approved by the
Animal Experimental Ethics Committee, Shanghai Ninth Peoples
Hospital affiliated to Shanghai Jiao Tong University School of
Medicine (SJTUSM) (Permit Number: HKDL 5).
Ta and Ti coatings were prepared by a VPS system (Sulzer,
Winterthur, Switzerland). Medical Ti-6Al-4V substrate was used
to prepare Ta-coated implants and interface samples. The samples
for investigating hBMSC proliferation and differentiation in vitro
were cuboid with dimensions of 10 mm610 mm62 mm and
cylindrical with dimensions of 33 mm62 mm. The samples for
implantation in vivo to examine osseointegration and new bone
formation rate were cylindrical with dimensions of
2 mm610 mm.
The coating surface was characterized using the following
equipment: an X-ray diffraction (XRD) instrument (D/max
2550v, Rigaku, Japan; MDI Jade 6.5), a contact angle analyzer
(SL200, Kino, Shanghai, P.R. China; with distilled water), a field
emission scanning electron microscopy (SEM) system (JSM 6700F,
JEOL, Akishima, Tokyo, Japan), and an electron probe
microanalyzer (EPMA, JXA-8100, JEOL, Akishima, Tokyo, Japan).
Isolation and Culture of hBMSCs
The hBMSCs were isolated and cultured as previously
described . A humidified 37uC/5% CO2 incubator
(MCO18AIC (UV), SAVYO, Panasonic, Kadoma, Osaka, Japan) was
used for cell culture with growth medium [a-MEM (Hyclone,
Thermo Fisher Scientific, Waltham, MA, USA) supplemented
with 10% FBS (Hyclone, Thermo Fisher Scientific, Waltham, MA,
USA), 100 U/mL penicillin, and 100 mg/L streptomycin
(Hyclone, Thermo Fisher Scientific, Waltham, MA, USA)].
The hBMSCs were seeded in six-well plates (Corning, Corning,
NY, USA) at a density of 56104 cells/well. After confluence in
growth medium, the cells were treated with osteogenic induction medium
[a-MEM supplemented with 10% FBS, 50 mM L-ascorbic acid
2phosphate (SigmaAldrich, St. Louis, MO, USA), 10 mM
bglycerophosphate (SigmaAldrich, St. Louis, MO, USA), 100 nM
dexamethasone (SigmaAldrich, St. Louis, MO, USA), 100 U/
mL penicillin, and 100 mg/L streptomycin], and then replaced
every 3 days. After 7 days, the cells were stained using a BCIP/
NBT ALP Color Development Kit (Beyotime, P.R. China). Cells
cultured in normal growth medium served as control.
The hBMSCs were seeded in six-well plates (Corning, Corning,
NY, USA) at a density of 26105 cells/well. After confluence in
growth medium, the cells were treated with adipogenic induction medium
[a-MEM supplemented with 10% FBS, 1 mM dexamethasone
phosphate, 200 mM Indomethin (SigmaAldrich, St. Louis, MO,
USA), 10 mg/mL insulin (SigmaAldrich, St. Louis, MO, USA),
0.5 mM 3-isobutyl-1-methylxanthine (IBMX, SigmaAldrich, St.
Louis, MO, USA), 100 U/mL penicillin, and 100 mg/L
streptomycin] for 2 days. Thereafter, adipogenic maintenance medium
[(aMEM supplemented with 10% FBS, 10 mg/mL insulin, 100 U/
mL penicillin, and 100 mg/L streptomycin)] was used and
replaced every day for 14 days. Oil Red O staining was performed
and then the sample was observed under a microscope (Nikon,
Shinjuku, Tokyo, Japan). Cells cultured in normal growth medium
served as control.
Cell Proliferation and Adhesion
The 10 mm610 mm62 mm samples were placed in 24-well
plates (Corning, Corning, NY, USA) and the hBMSCs were
seeded at 26104 cells/well with the growth medium. After 24 h, the
samples were transferred to new 24-well plates for further
experiment. DRAQ5 (Danvers, MA, USA) was used as fluorescent
DNA dye. The samples were scanned with an Odyssey
nearinfrared scanner (LI-COR Biosciences, Millipore, Billerica, MA,
USA) to determine the proliferation rate variation of hBMSCs on
porous Ta and Ti coating. PrestoBlue Cell Viability Reagent
(Invitrogen, Life Technologies, Carlsbad, CA, USA) was used
according to the manufacturers instructions to test cell viability,
and the absorbance was obtained using a microplate reader
(Infinite M200 pro, TECAN, Switzerland) at wavelengths of 570
and 650 nm. LIVE/DEAD Cell Viability Assays (Invitrogen, Life
Technologies, Carlsbad, CA, USA) were performed as described
in the manual. F-actin was identified by fluorescent phallotoxin
(Phalloidin-TRITC, SigmaAldrich, St. Louis, MO, USA).
AntiKi67 antibody (Abcam, Cambridge, UK) was used to detect Ki-67
protein expression in hBMSCs, and DAPI (SigmaAldrich, St.
Louis, MO, USA) was used to indicate the nucleus of hBMSCs.
LIVE/DEAD Cell Viability Assays, fluorescent phallotoxin, and
anti-Ki67 antibody/DAPI were observed by confocal laser
scanning microscopy (LSM510, Carl Zeiss AG, Oberkochen,
The CFSE labeling experiment was carried out as described
[18,19]. CFDA-SE (SigmaAldrich, St. Louis, MO, USA) was
used for labeling. The CFSE labeled hBMSCs were seeded on the
33 mm62 mm samples which placed in six-well plates
(Corning, Corning, NY, USA). After 24 h, the samples were
transferred to new six-well plates. The hBMSCs with CFSE
labeling were cultured for total 7 days and then analyzed by flow
cytometers FACS Aria (Becton Dickinson, Franklin Lakes, NJ,
ALP Staining of hBMSCs on Coatings
The 32 mm62 mm samples with coating were placed in
sixwell plates (Corning, Corning, NY, USA) and the hBMSCs were
seeded at 56104 cells/well with the growth medium. After 24 h, the
samples were transferred to new six-well plates, and after another
24 h, the growth medium was replaced with the osteogenic induction
medium, which was replaced every 3 days. The cells were stained
using a BCIP/NBT ALP Color Development Kit (Beyotime, P.R.
China), and then scanned with an Odyssey near-infrared scanner
(LI-COR Biosciences, Millipore, Billerica, MA, USA) and a
normal scanner (Cannon, Tokyo, Japan).
plate serviced as calibrator). (C) LIVE/DEAD Cell Viability Assays of hBMSCs on the surface of the coatings in 48 h. (D) Ki67 and DAPI stains of hBMSCs
on the surface of the coatings in 48 h. (E) The flow cytometric analyses of hBMSC with CFSE on the surface of the coatings on day 7.
In-Cell Western assay was performed following the protocol of
CST Ltd. to evaluate the protein expression of ALP in hBMSCs
on porous Ta coatings. Paraformaldehyde (4%) was used for
fixation. Odyssey Blocking Buffer (LI-COR Biosciences, Millipore,
Billerica, MA, USA) was used as a blocking and antibody-diluting
buffer. ALP antibody (Santa Cruz Biotechnology, Santa Cruz,
CA, USA) was used to detect the protein expression of ALP in
hBMSCs on the coatings. After incubation, Infra-Red Secondary
Antibody IRDye (Rockland Immunochemicals, Gilbertsville, PA,
USA) was used as a secondary antibody and DNA staining dye
DRAQ5 (Danvers, MA, USA) was used as normalizing agent .
The result was acquired by an Odyssey Infrared Imaging System
(LI-COR Biosciences, Millipore, Billerica, MA, USA).
Total RNA of cells was isolated using TriPure Isolation Reagent
(Roche, Basel, Switzerland) according to the manufacturers
instructions. Equivalent amount of RNA samples were reverse
transcribed for first-strand cDNA using a RevertAid First Strand
cDNA Synthesis Kit (Fermentas, Thermo Fisher Scientific,
Waltham, MA, USA). Real-time PCR was performed by
Lightcycler480 (Roche, Basel, Switzerland) using SYBR Premix Ex
TaqTM (Takara, Otsu, Shiga, Japan) according to the
manufacturers instructions. GAPDH was used as a reference. The
realtime PCR conditions were as follows: denaturation at 95uC for
10 s, 40 cycles at 95uC for 10 s, and 60uC for 20 s. Dissociation
was performed for a melting curve analysis to monitor and avoid
non-specific amplification as well as primer dimers. The collected
data were analyzed by the advanced relative quantification
method using Roche Lightcycler480 software.
Animals and Implantation
New Zealand white rabbits (male, 3 months old, 2.83.0 kg)
were used as the femur implant model. The operation was
performed under aseptic conditions by chloral hydrate
intramuscular injection. Defect in each femoral condyle was made by a
2 mm drill toward the medial epicondyle orientated perpendicular
to the longitudinal and sagittal axes . After placing the implant
in the hole, incisions were closed with sutures. To avoid wound
infection, each animal was given an intramuscular injection of 400
000 U penicillin per day for 3 days after operation.
Double Fluorescence Labeling and Harvesting of Bone
The first labeling was performed by intravenous injection
10 days before sacrificing the rabbits, and the second labeling was
performed 3 days before sacrificing. Calcein (8 mg/kg, Sigma
Aldrich, St. Louis, MO, USA) was used for both labelings.
After cleaning the soft tissue, the samples were fixed in 4%
paraformaldehyde buffer in PBS for 10 days and then dehydrated
in successive alcohol concentrations (70%, 75%, 80%, 85%, 90%,
95%, and 100% per day). The dehydrated sample was embedded
in Technovit 7200VLC (Exakt, Norderstedt, Hamburg, Germany)
for 2 days using the EXAKT 510 Dehydration and Infiltration
System (Exakt, Norderstedt, Hamburg, Germany), and then
polymerized by the EXAKT 520 Light Polymerization System
(Exakt, Norderstedt, Hamburg, Germany). Subsequently, 300 mm
sections were cut perpendicular to the implants using Saw
Microtome Leica SP1600 (Leica, Wetzlar, Germany), ground to
50 mm by the EXAKT 400 CS Micro Grinding System with AW
110 controller (Exakt, Norderstedt, Hamburg, Germany), and
finally stained by Van Giesons PicricFuchsin stain for
Different histomorphometric parameters were obtained using a
microscope (LEICA DM 4000B, German). A semiautomatic
image analysis system (BIOQUANT) was used to measure the
surfaces of the sections. The area of 50 pixels around the implant
was selected as the region of interest (ROI), amounts of bone
tissues in the ROI were calculated by using the measurement tools.
Bone volume fractions (ratio of bone tissue area to the total area in
the ROI) of different samples were calculated based on Van
Giesons PicricFuchsin staining and compared statistically.
Statistical analysis was performed using SAS JMP (Cary, NC,
USA). Aspin-Welch-Satterthwaite-Students t-test (t-test works
even if variances in the two sample groups are different) were
used to compare the means of the two sample groups.
Identification of hBMSCs
The hBMSCs were isolated as previously described , and
then used to evaluate the biocompatibility and osteoinductivity of
porous Ta and Ti coatings. The isolated cells expressed the MSC
markers CD29, CD105, CD44 and CD90, but were negative for
the hematopoietic marker CD34 and leukocyte marker CD45
(Figure 1A). These findings were similar to the hBMSC
population phenotypic characteristics described in previous
research . The differentiation potential of hBMSCs was
also investigated by osteogenic and adipogenic induction, followed
by ALP and Oil Red O staining. The results demonstrated the
hBMSCs potential to differentiate into the lineages of osteoblast
and adipocyte (Figure 1B).
Surface Morphology of Coatings
The XRD results showed that the Ti coating surface was mainly
composed of Ti, and the composition of the Ta coating was major
Ta and a minor Ta oxide (Figure 2A). The oxide layer of Ta
(Ta2O5) formed on the surface (Figure 2A) was quite stable at
various pH values [2,5]. The contact angle of porous Ta coating
surface insignificantly differed from that of the Ti coating
(Figure 2B) (n = 3; Ti coating, 131.91765.10882; Ta coating,
130.45565.55294; P = 0.7125). The initial surface morphology of
Ta and Ti coatings was observed by SME at 3006, 5006, 10006,
and 30006 magnifications (Figure 2C), showing that the surface
roughness of both Ta and Ti coatings was similar.
Cell Shape and Cytoskeletal Tension of hBMSCs on Ta
The hBMSCs displayed much better expanding on the porous
Ta coating surface compared with the Ti coating control and the
uncoated control (Figure 3A and B). In Figure 3A, the SME
Figure 5. ALP and RUNX2 expression of hBMSCs on Ta and Ti coatings with osteogenic induction. (A) Odyssey scanning of the ALP
protein expression in hBMSCs on the coatings with DRAQ5 as reference, 12 days. (B) ALP activity stain assay of hBMSCs on the coatings in 3, 7, 15,
and 21 days with a white light scanner and near-infrared Odyssey scanner. (C) Real-time PCR detection of the mRNA expression of RUNX2 gene in
hBMSCs on the coatings with osteogenic induction for 21 days. GAPDH was used as reference gene (t-test, assuming unequal variances, n = 3; Ti
coating, 160.1404; Ta coating, 1.967860.2370; Prob.|t|, ,0.01*).
Figure 6. New bone formation around porous Ta and Ti coating implants in vivo. (A) Van Giesons PicricFuchsin stain of new bone
formation around implants. Red indicates the bone around implants in 3 months. (B) Quantification of new bone formation around the coating
implants in 3 months in vivo (t-test, assuming unequal variances, error bars represent 6 SD, n = 3; Ti coating, 14.074166.46293; Ta coating,
32.650160.90721; Prob.|t|, 0.0358*). (C) Double fluorescence labeling was used to represent the mineral apposition rate of new bone formation.
Green with white arrow indicates the calcein fluorescence labels in 3 months.
image showed that the hBMSCs on Ta coating had a flattened
expanded shape. F-actin cytoskeletal staining (Figure 3B)
indicated the different cytoskeletal tensions and the flattened expanded
shape of hBMSCs on Ta coating compared with the contracted
shape on Ti coating.
Adhesion and Proliferation of hBMSCs on Ta Coatings
An immunofluorescence experiment was conducted in which
the DNA dye Draq5 was applied to monitor cell number (density).
The result (Figure 4A) showed that the hBMSCs proliferated
much faster on porous Ta than on Ti coating in 48 h (n = 3; Ti
coating, 65.377618.2972; Ta coating, 164.80646.1212;
P,0.0498*). In Figure 4B, the data of PrestoBlue assay showed
that the hBMSCs had a significantly faster proliferation on the
porous Ta coating surface than on the Ti coating control after 4
and 6 days (n = 3). For day 2, the data were as follows: Ti coating,
0.4526860.097954; Ta coating, 1.076760.495686; P,0.992. For
day 3, the data were as follows: Ti coating, 0.5695060.089554; Ta
coating, 1.2622860.458399; P,0.0620. For day 4, the data were
as follows: Ti coating, 0.9027560.082739; Ta coating,
2.1323960.699593; P,0.0390*. For day 6, the data were as
follows: Ti coating, 0.8675260.089092; Ta coating
1.6868860.386935; P,0.0233*. LIVE/DEAD Cell Viability
assay and anti-Ki67 immunofluorescence assay (Figures 4C
and 4D, respectively) demonstrated not only faster proliferation
but also less cell death of hBMSCs on porous Ta coating than on
Ti coating in 48 h. The CFSE labeling results (Figure 4E) also
showed hBMSCs on Ta coating surface had a higher proliferation
rate than those on Ti coating surface.
ALP and RUNX2 Expression in hBMSCs on Ta Coatings
The hBMSCs were cultured on porous Ta and Ti coatings with
the osteogenic medium, and then ALP and RUNX2 were detected
as osteoplastic differentiation-related markers. Higher ALP activity
and expression were detected (Figures 5A and 5B) on porous Ta
coating by immunofluorescence detection of ALP with DRAQ5 as
reference (12 days, 0.057 vs 0.029 with Odyssey calculation) and
ALP staining (7, 15, and 21 days). Real-time RT-PCR was used to
detect the mRNA expression of RUNX2 when the hBMSCs were
cultured on the porous Ta coating and on the porous Ti coating
control in osteogenic medium after 21 days. The expression of
RUNX2 was significantly enhanced in hBMSCs cultured on
porous Ta coating compared with Ti control coating (Figure 5C)
(n = 3; Ti coating, 160.1404; Ta coating, 1.967860.2370;
P,0.01*). These results suggested that the porous Ta coating
may be more beneficial for hBMSC osteogenesis than Ti coating.
Osseointegration of Implants with Porous Ta Coating
Van Giesons PicricFuchsin stain of transverse sections was
used to show the osseointegration of the porous Ta and Ti
coatings. The mineral apposition rate of new bone formation was
also investigated by measuring the distances of double fluorescence
labeling. After implantation for 3 months, histomorphometric
parameters clearly indicated that more new bone was formed
around the porous Ta coating implant than around the Ti coating
implant (Figures 6A and 6B) (n = 3; Ti coating,
14.074166.46293; Ta coating, 32.650160.90721; P,0.0358*).
The double fluorescence labeling (Figure 6C) also showed that
the mineral apposition rate of new bone formation was higher in
the group implanted with porous Ta coating.
Over the last two decades, a variety of porous coatings and
materials have been used to achieve biological fixation of implants.
Ti alloys are found to be very suitable materials for load-bearing in
bioimplant applications because of their good and reliable
mechanical properties. Unfortunately, like most metals, Ti exhibits
poor osteoinductive properties. Among metallic biomaterials, Ta is
gaining increased attention as a new biomaterial. Ta has been
demonstrated to be corrosion resistant and bioactive in vivo.
Although Ta has been used for plates and suture wires in
orthopedic, craniofacial surgery, and dentistry , the relatively
high manufacturing cost and difficulty in fabrication have limited
its widespread acceptance. To utilize fully the biocompatibility of
Ta for load-bearing metal implants, in this work, porous Ta
coating were prepared with Ti as substrate. The biocompatibility
and osteoinductivity of Ta coatings applied by VPS were then
The in vitro hBMSC-material interaction results clearly
indicated that the porous Ta coating surface significantly improved the
adhesion and proliferation of hBMSCs, as assessed by SEM cell
morphology, F-actin cytoskeletal staining, immunofluorescence,
and PrestoBlue assay. The characteristics and biocompatibility of
implant surface material were closely related to cell-material
interactions, and can influence cell response and behavior [25,26].
Our results showed the superior cell adhesion and proliferation, as
well as less cell death of hBMSCs on the porous Ta coating
compared with Ti coating. These findings indicated that Ta
surfaces were biocompatible and significantly influenced hBMSC
biological behavior. Generally, the initial adhesion influences the
subsequent differentiation of stem cells [3,2736]. The different
cytoskeleton signals from adhesion are transduced to the nucleus,
where they can result in a change in gene expression and,
subsequently, differentiation [36,37]. McBeath et al.  proved
that the cell shape regulates hBMSCs commitment to osteoblast
or adipocyte fate (i.e., hBMSCs allowed to adhere, flatten, and
expanded undergo osteogenesis, whereas condensed, round cells
become adipocytes), and that F-actin cytoskeletal is involved in this
process. In this study, F-actin cytoskeletal staining reveals the
flattened expanded shape of hBMSCs on Ta coating compared
with the contracted shape on Ti coating. Therefore, the difference
in hBMSCs F-actin cytoskeleton on porous Ta and Ti coatings
partly explained the higher osteogenesis of hBMCs on the porous
Ta coating implants.
Moreover, the porous Ta coating not only enhanced hBMSC
adhesion and proliferation, but also stimulated mesenchymal stem
cell osteogenetic differentiation in vitro compared with porous Ti.
This finding was confirmed by the enhanced ALP and RUNX2
activity on Ta coating. The possible explanation was that the
bone-like elasticity substrates may have been better for bone
regeneration and contributed to the differentiation lineage of the
hBMSCs [39,40]. Thus, given its similar elasticity to cancellous
bone, porous Ta coating was more beneficial for osteogenesis. In
vivo, which used a rabbit femur implant model, we found that the
implants coated with Ta had a higher rate of osseointegration than
the Ti coating implants because of the excellent biocompatibility
and bioactivity of Ta. These key properties of porous Ta can be
attributed to its ability to form a self-passivating surface oxide
layer. This surface layer leads to the formation of a bone-like
apatite coating in vivo, ensuring excellent bone and fibrous
ingrowth properties for rapid and substantial bonesoft tissue
attachment . Our in vivo results on the accelerated rate of
new bone formation on Ta coating implant compared with Ti
coating implant well agreed with in vitro studies demonstrating
enhanced stem cell adhesion, proliferation, and osteogenic
differentiation on a Ta coating surface over a Ti coating surface.
These results revealed the ability of porous Ta coating to
support the differentiation of mesenchymal stem cells into
osteoblasts in vitro and of matured bone cells in vivo. Porous Ta
was found to have excellent biocompatibility and be safe to use
in vivo . Core decompression with porous Ta implants has
shown encouraging success rates in early clinical results among
patients with advanced stage osteonecrosis . Thus, porous Ta is
indeed an attractive option for clinical applications. Although
long-term experimental and clinical studies are required to verify
the advantages and outcomes of such an implant, a novel
application of porous Ta as an orthopedic implant coating applied
by VPS for promoting bone regeneration was presented.
Porous Ta coatings were fabricated by VPS with Ti alloy as
substrate. The deposition of porous Ta layer effectively alleviated
the mechanical incompatibility between metal implant and bone
tissue. The biocompatibility, osteoinductivity, and osseointegration
of the Ta coated implants were studied to determine their
feasibility in clinical applications. In vitro, hBMSCs were used and
the results showed that hBMSC adhesion, proliferation, and
osteogenic differentiation were enhanced on the Ta coating
surface compared with the Ti coating control. Moreover, in vivo
implantation in a rabbit femur defect model confirmed the
excellent osseointegration and new bone formation of the porous
Ta coated implants. These results suggested that porous Ta
coating applied by VPS is a promising strategy for bone
Conceived and designed the experiments: YX KD X. Zheng X. Zhang.
Performed the experiments: ZT YX FY CW. Analyzed the data: ZT YH.
Wrote the paper: ZT YH X. Zhang.
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