polymers
ArticleFluorescent Nanodiamonds Enable Long-Term
Detection of Human Adipose-Derived Stem/Stromal
Cells in an In Vivo Chondrogenesis Model Using
Decellularized Extracellular Matrices and Fibrin
Glue Polymer
Yi-Chia Wu 1,2,3,4,5 , Ya-Chin Wang 3 , Wei-Ting Wang 5 , Hui-Min David Wang 3,6 ,Hsin-Hung Lin 7 , Long-Jyun Su 7 , Yur-Ren Kuo 3,4 , Chung-Sheng Lai 4 , Mei-Ling Ho 3,8and John Yu 1,9, *
1
2
3
4
5
6
7
8
9

*

Institute of Stem Cell and Translational Cancer Research, Chang Gung Memorial Hospital at Linko,
Taoyuan 333, Taiwan
Ph.D. Program in Translational Medicine, Kaohsiung Medical University, Kaohsiung, and Academia Sinica,
Taipei 115, Taiwan
Regeneration Medicine and Cell Therapy Research Center, Kaohsiung Medical University,
Kaohsiung 807 Taiwan
Division of Plastic Surgery, Department of Surgery, Kaohsiung Medical University Hospital,
Kaohsiung 807, Taiwan
Center of Teaching and Research, Kaohsiung Municipal Ta-Tung Hospital, Kaohsiung 801, Taiwan
Graduate Institute of Biomedical Engineering, National Chung Hsing University, Taichung 402, Taiwan
Institute of Atomic and Molecular Sciences, Academia Sinica, Taipei 106, Taiwan
Orthopaedic Research Center, Kaohsiung Medical University, Kaohsiung 801, Taiwan
Institute of Cellular and Organismic Biology, Academia Sinica, Taipei 115, Taiwan
Correspondence: johnyu@gate.sinica.edu.tw; Tel.: +886-3-328-1200#7803

Received: 16 June 2019; Accepted: 16 August 2019; Published: 23 August 2019


Abstract: Clinically available materials, including allogeneic irradiated costal cartilage and fibrin glue
polymer, were used as scaffolds for in vivo chondrogenic differentiation of human adipose-derived
stem/stromal cells (hASCs) in the attempt to develop a more efficient treatment over current methods.
Current studies include the use of growth-factor stimulation, tissue engineering, and biocompatible
materials; however, most methods involve complicated processes and pose clinical limitations.
In this report, the xenografts in the experimental group composed of a diced decellularized cartilage
extracellular matrix (ECM), hASCs, and fibrin glue polymer were implanted into the subcutaneous
layer of nude mice, and the results were compared with two groups of controls; one control group
received implantation of decellularized cartilage ECM and fibrin glue polymer, and the other control
group received implantation of hASCs mixed with fibrin glue polymer. To evaluate whether hASCs
had in vivo chondrogenesis in the xenografts, hASCs were labeled with fluorescent nanodiamonds(FNDs), a biocompatible and photostable nanomaterial, to allow for long-term detection andhistological analysis. Increased cellularity, glycosaminoglycan, and collagen deposition were foundby the histological examination in the experimental group compared with control groups. With thebackground-free detection technique and time-gated fluorescence imaging, the numbers and locationsof the FND-labeled hASCs could be detected by confocal microscopy. The chondrocyte-specificmarkers, such as aggrecan and type II collagen, were colocalized with cells containing signals of FNDs
which indicated in vivo chondrogenesis of hASCs. Taken together, functional in vivo chondrogenesisof the hASCs could be achieved by clinically available decellularized cartilage ECM and fibrin glue
polymer in the nude mice model without in vitro chondrogenic induction. The fluorescent signals of
FNDs in hASCs can be detected in histological analysis, such as hematoxylin and eosin staining (H&E

Polymers 2019, 11, 1391; doi:10.3390/polym11091391

www.mdpi.com/journal/polymers

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staining) without the interference of the autofluorescence. Our study may warrant future clinicalapplications of the combination of decellular cartilage ECM, fibrin glue polymer, and hASCs for
cartilage repair.
Keywords: human adipose-derived stem/stromal cells (hASCs); in vivo chondrogenesis; decellularized
cartilage ECM; fibrin glue polymer; fluorescent nanodiamonds (FNDs); long-term detection

1. Introduction
Articular cartilage is a highly specialized tissue acting as an absorber of weight during sustained
static loading that cushions the ends of bones where they come together to form joints [1]. As anavascular tissue with low cellular biosynthetic activity, cartilage has limited repair potential [2,3].
Articular cartilage damage from trauma or degenerative pathology often results in progressive
deterioration, leading to joint pain, functional impairment, and degenerative arthritis [1,2]. Surgicalinterventions, aiming to either replace damaged chondral tissue or to induce repair, include
arthroscopic debridement [4], bone marrow stimulation techniques [5,6], chondrocyte implantation [7,8],
osteochondral autografts [9], osteoarthritis allograft [10], and total joint replacement for end-stage
degenerative joint pathology [11].
Many of these approaches, however, often result in the formation of fibrocartilage that isbiochemically and biomechanically inferior to hyaline articular cartilage. Autologous chondrocyte
implantation and its later relevant approach, matrix-induced autologous chondrocyte implantation,offer great promise in clinical and histological results [12,13], but many adverse complications havebeen reported, including graft failure, delamination, and tissue hypertrophy [14,15]. In addition,
the ability of autologous chondrocytes to regenerate cartilage neotissues decreases with patients’ age
and in vitro expansion [16], making them a substandard cell source for cartilage repair. The repair ofcartilaginous lesions is still a weighty clinical problem.
Development of stem-cell-based therapies is one of the most promising advancements in celltherapy and regenerative medicine. Mesenchymal stem/stromal cells (MSCs) derived from tissues suchas bone marrow, adipose, or placenta are defined as self-renewal, multipotent progenitor cells with theability to differentiate into multiple lineages, such as adipocytes, chondrocytes, and osteocytes [17].
MSCs are one of the ideal sources for stem-cell-based therapies due to their multilineage differentiation
potential and immunomodulatory properties [18]. hASC is an ideal source for cartilage-tissue
engineering, owing to their accessibility, capacity for self-renewal, and potential to synthesize
cartilage-specific matrix proteins that are assembled in a cartilage ECM [19,20].
Various studies have reported that tissue engineering of cartilage has been targeting on scaffolds,
cells, and addition of growth factors [21,22]. Allogenic costal cartilage may be used as a decellularized
cartilage ECM for the surgical reconstruction of damaged cartilaginous tissue. The combination of
decellularized cartilage ECM, hASCs, and fibrin glue polymer for cartilage regeneration could solve
several shortcomings, such as donor site morbidity, limited cell sources, and multiple surgeries [3].
In addition, fibrin has been widely applied as a scaffold in clinical purposes and is one of the suitablescaffolds for chondrogenic differentiation of stem/stromal cells [23,24]. Fibrin scaffold provides auseful platform and appropriate environment for chondrogenesis of hASCs [25] because of its adhesive
properties, biocompatibility, and biodegradation [26]. TISSEEL is mainly composed of fibrinogen(85 mg/mL) and thrombin (500 IU/mL), which are mixed in a 1:1 ratio in the course of application.
The main protein components of finial fibrin glue polymers are fibrin, fibronectin, and albumin.
The fibrin concentrations within the clots are 45 mg/mL. It polymerizes on its own and was approvedby the U.S. Food and Drug Administration for hemostats, sealants, and adhesives [27,28]. The hASCs
were adhesive to the decellularized cartilage ECM to form the xenograft constructs in our experiments.

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Taken together, a fibrin scaffold composed of clinically available decellularized cartilage ECM andfibrin glue polymer could be an ideal material for in vivo chondrogenesis of hASCs.
Accurate stem-cell tracking in vivo is one of the most important requirements in regenerative
medicine. Cell tracking determines both stem cell destinations and final differentiation fates, thus
allowing a more detailed picture of the mechanisms involved in these therapies [21]. In order toinvestigate the efficacies and safety throughout the course of stem-cell-based treatment, it is vitalto develop a method for tracking the stem cells. [22]. At present, long-term tracking of cells is
one of the most difficult hurdles to overcome for studying in vivo chondrogenesis. Fluorescent
nanodiamonds (FNDs) [29] could be an ideal long-term cell tracking device for studying in vivochondrogenesis. FND is a carbon-based nanomaterial [30] with extraordinary chemical stability andbiological inertness [29,30]. A number of studies have demonstrated that FNDs can be internalized
by cells through clathrin-mediated endocytosis [31] with low exocytosis activity [32,33]. FNDs
can be widely used for biomedical research as a novel nanotechnology for image probing [34,35].
The negatively charged nitrogen-vacancy (NV− ) color centers in the diamond lattice of FNDs as
built-in fluorophores which have perfect photostability, without photobleaching and blinking [29].
When the greenish-yellow light excites the NV− centers of FNDs by a laser, the NV− centers emits
stable far-red emission (around 600–800 nm) with the fluorescence lifetime (τ) being longer than 15 ns,
which can avoid the interference of a strong autofluorescence background from the host tissue using
time-gating techniques [32]. Taking advantage of the unique magneto-optical property of NV− centers,
the absolute number of transplanted cells can be quantified by magnetically modulated fluorescence(MMF), a background-free detection method [34].
In this work, hASCs labeled with FNDs had unaffected cellular functions, such as proliferationand differentiation. The xenografts of the experimental group composed of the decellularized cartilage
ECM, fibrin glue polymer, and hASCs were implanted into nude mice. FND-labeled hASCs were clearly
identified and quantified in xenografts three months afterwards. The xenografts of the experimentalgroup had increased cellularity and volume compared with the control groups. Furthermore, increased
glycosaminoglycans and collagen were also observed. The chondrocyte-specific markers, such as
aggrecan and type II collagen, were colocalized with the signals of FNDs, which might indicate in vivochondrogenesis of hASCs. The FNDs are applicable for long-term detection of stem/stromal cells whichmight undergo cell differentiation.
2. Materials and Methods
2.1. FNDs Production and Characterization
Synthetic type Ib diamond powders (Micron + MDA, Element Six, London, UK) with a mean sizein the range of ~100 nm were radiation-damaged by ion bombardment with 40-keV He+, followedby annealing in vacuum at 800 ◦ C, oxidization in air at 490 ◦ C, and purification in concentrated
H2 SO4 –HNO3 (3:1, v/v) at 100 ◦ C, as previously described [35]. The size and spectrum of FNDs were
determined by Fluorescence spectrometer and dynamic light scattering. Normalized fluorescence
spectrum of FNDs and human serum albumin (HSA)-conjugated FNDs (FND@HSA) in water (1 mg/mL)
were acquired by laser excitation at 532 nm. Size distributions of the particles in both distilled deionizedwater and PBS were measured with a combined particle size and zeta potential analyzer (Delsa Nano
C, Beckman-Coulter, Brea, CA, USA).
2.2. Culture of Human Adipose-Derived Stem/Stromal Cells (hASCs)
The hASCs were purchased from Lonza (PT-5006, Bazel, Switzerland) and cultured with complete
K-medium [Defined Keratinocyte-SFM (Thermo Fisher Scientific, Waltham, MA, USA) supplemented
with 5% FBS (Thermo Fisher Scientific, Waltham, MA, USA), 1% Antibiotic-Antimycotic (Thermo Fisher
Scientific, Waltham, MA, USA), 57.8 µg/mL L—Ascorbic acid, 2-phosphate sesquimagnesium saltPolymers 2019, 11, 1391

4 of 16hydrate (Sigma-Aldrich, St. Louis, MO, USA), and 326.4 µg/mL N—Acetyl-L-cysteine (Sigma-Aldrich,
St. Louis, MO, USA) in a humidified incubator at 37 ◦ C with 5% CO2 .
2.3. Cell Labelling
Prior to cell labeling, 100 nm FNDs were first dispersed in serum-free DMEM and then diluted
with the medium to have a concentration in the range of 5–200 µg/mL, as indicated. hASCs were plated
in culture dishes and labeled with FNDs in serum-free medium at 37 ◦ C with 5% CO2 for four hours,
and then thoroughly washed with PBS to remove free FNDs by centrifugal separation.
2.4. MTT Assay
1 × 105 hASCs were seeded into 6-well culture dishes overnight to allow cell attachment before
FNDs treatment. Afterwards, cells were refreshed with medium containing 10 µg/mL MTT under 37 ◦ C,
5% CO2 in the dark for two hours. Dimethyl sulfoxide (DMSO) was used to dissolve formazan crystals
produced by living cells. Cell viability was determined and quantified by measuring absorbance at awavelength of OD 570.
2.5. Flow Cytometry Analysis
Single-cell suspensions of hASCs were stained. Aliquots of 1 × 104 cells were labeled withfluorescein isothiocyanate (FITC)—conjugated monoclonal antibodies against human CD29, CD31,
CD34, CD44, CD45, and CD90 for 30 min at 4 ◦ C. The cells were washed three times in cold PBS andanalyzed using a BD LSR II (BD Biosciences, San Jose, CA, USA). Antibodies recognizing CD29, CD31,
CD34, CD44, CD45, and CD90 were purchased from Abcam (Cambridge, UK).
2.6. In Vitro Multipotency Differentiation Assay
The hASCs, no later than passage three, were plated into 6-well plates at a density of 106 cells/welland treated with 100 µg/mL FNDs. The cells were then induced to adipogenic, osteogenic, orchondrogenic differentiation by using hMSC adipogenic induction SingleQuots™ kit (Lonza PT-4135,
Bazel, Switzerland) or a hMSC osteogenic SingleQuots™ kit (Lonza PT-4120, Bazel, Switzerland) or
Human Mesenchymal Stem Cell Chondrogenic Differentiation Medium BulletKitTM (Lonza PT-3003,
Bazel, Switzerland) following the instruction manual provided with the kit. These differentiatedlineages derived from hASCs were further confirmed by Oil Red O (adipogenesis) or Alizarin Red(osteogenesis) or Alcian Blue (chondrogenesis) staining, respectively.
2.7. Animals and In Vivo Implantation
Thirty nude male mice purchased from the National Laboratory Animal Center at five weeks
of age were acclimatized for up to six weeks prior to use. The animal protocol was followed andapproved by the Kaohsiung Medical University Animal Care Advisory Committee (No. 106056).
Mice were housed and cared for at the animal facility of Kaohsiung Medical University. The mice wererandomly allocated into three groups (ten mice/group): (1) the ECM-glue group, composed of diced
decellularized cartilage ECM and fibrin glue polymer (TISSEEL Solution; Baxter AG; Vienna, Austria);(2) the ECM-hASC-glue group, composed of 1 × 105 hASCs in diced decellularized cartilage ECM andfibrin glue; or (3) the hASC-glue group, composed of 1 × 105 hASCs in fibrin glue polymer.
2.8. Xenograft Construct Preparation and Measurement
We grounded the allogeneic irradiated costal cartilage (LifeNet, Virginia Beach, VA, USA) into
particles approximately 0.5 mm × 0.5 mm × 0.5 mm in size as decellularized cartilage ECM in thisstudy. The grounded cartilages were loaded into a syringe (Figure S3a). The fibrin glue polymers, withor without hASCs, were then loaded into the syringe (Figure S3b). The volume of each xenograft was
0.5 mL. The xenografts were implanted subcutaneously into six-week nude mice for three months.

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Three months later, the xenografts were retrieved and placed in a syringe, and injected into the syringewith PBS to a volume of 1 mL. The xenograft was taken out, and the volume of the PBS was thenrecorded. The xenografts were then subjected for histological analysis and MMF.
2.9. Histological Analysis
The harvested xenografts were fixed in 4% paraformaldehyde (Sigma, St. Louis, MO, USA)overnight and stored in 70% ethanol at 4 ◦ C for further processing. Xenografts were then embedded inparaffin and processed following the standard histological procedures. The xenograft structures weresliced into sections which were 5 µm thick. Sections were stained with hematoxylin and eosin.
2.10. Quantification of Cell Number
The harvested xenografts were sliced into sections which were 5 µm thick. Each section was
mounted with fluorescence mounting medium, including 40 ,6-diamidino-2-phenylindole (DAPI)(ab104139, Abcam, Cambridge, MA, USA) for counterstaining. The image of the section was taken
under a fluorescence microscope with 20-fold magnification. The cell numbers in six random fields ofeach section were counted.
2.11. Alcian Blue Staining
The sections were fixed with 3.7% formaldehyde in PBS for 30 min, and then rinsed with ddH2 O;
then, they were stained with Alcian blue (pH 2.5, Muto Pure Chemicals, Tokyo, Japan) for 60 min.
Afterwards, cells were washed with 3% acetic acid for three minutes to remove nonspecific staining
and washing with PBS.
2.12. Masson’s Trichrome Staining
The sections were stained with Masson’s Trichrome staining by following the protocol of Masson’s
Trichrome staining kit (Bio-Optica, Milano, Italy). The Zeiss Axiophot microscope (Carl Zeiss AG,
Werk Göttingen, Germany) was used to observe illumination through a bright light, and photographs
were acquired by a Discovery C30 camera (Tucsen Imaging Technology Co., Ltd. Fujian, China).
2.13. Immunofluorescence
The sections which were 5 µm thick were incubated in 0.1% trypsin (Gibco, ThermoFisher,Carlsbad, CA, USA) at 37 ◦ C for 15 min for enzymatic antigen retrieval. The sections were then blockedwith blocking buffer consisting of 5% milk in PBS, followed by incubation in a rabbit polyclonal antibody
to collagen type II (diluted 1:200, Abcam, Cambridge, MA, USA) or a goat monoclonal antibody toaggrecan (diluted 1:200) overnight at 4 ◦ C, and then incubation of a secondary antibody (diluted 1:200,
Alexa Fluor 488 goat anti-rabbit, ThermoFisher, Carlsbad, CA, USA) for an hour at room temperature.
Nuclei were stained with DAPI mounting gel (Vectashield, Vector Laboratories, Burlingame, CA, USA)to identify cellular nuclei. The images were taken with a Zeiss fluorescence microscope.
2.14. Magnetically Modulated Fluorescence (MMF)
Fluorescence spectra of FNDs suspended in aqueous solution were acquired by using a MMF
spectrometer built in-house [34]. The spectrometer was equipped with a continuous-wave 532 nm
laser (DPGL-2100F, Photop Suwtech, Shanghai, China), a dichroic beam splitter (Z532RDC, Chroma,
Taoyuan, Taiwan), a long-working distance microscope objective (50Å~, NA 0.55, Mitutoyo, Kanagawa
Prefecture, Japan), a long-pass edge filter (E550LP, Chroma, Taoyuan, Taiwan), and a multichannel
analyzer (C7473, Hamamatsu, Japan). Backward fluorescence was collected through the same objective
to avoid spectral distortion due to strong light-scattering by FNDs. To eliminate background noise,
we magnetically modulated the FND fluorescence signal, and then applied fast Fourier Transform (FFT)to extract concentration information from the measured fluorescence intensities. This was achieved by

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applying a time-varying magnetic field of 20 mT from a round electromagnet (EM400-12-212, APW,
Kuala Lumpur, Malaysia) to the sample solution held in a 10 mm cuvette with a frequency of 2 Hz.
For every wavelength, we obtained a time-domain spectrum, with the magnetic field periodicallyswitching on and off. A MATLAB program analyzed the time evolution of the spectra by performing
FFT to yield intensities at the modulation frequency. The FND spectra were finally restored by plotting
the demodulated fluorescence intensities against the wavelength. To conduct the analysis, the cells werefirst sonicated in a cuvette for 1 h to break up the cells. The amounts of the internalized FNDs in thecells were then determined from the fluorescence intensities against a calibration curve. To enable FND
quantification in the xenografts by the MMF method, we digested the xenografts in aqua regia/H2 O2
mixtures to release the nanoparticles into the solution. Fluorescence intensities were then measured
directly for FNDs in the tissue digests without extraction or other separation procedures to avoid lossof the particles during centrifugation or filtration. An important thing to note is that aqua regia is a
mixture of nitric acid and hydrochloric acid with an optimal molar ratio of 1:3. Regarding chemicalsafety, it is important to add the nitric acid to the hydrochloric acid during preparation. Since the acidmixture is unstable, it should be prepared in a small amount and used immediately.
2.15. Statistical Analysis
Results of multiple observations are presented as means ± S.E.M. For analysis of multivariate data,group differences were assessed using T tests, one-way or two-way ANOVA, followed by Turkey’s
multiple comparison tests by using GraphPad Prism version 7.00 for Windows (GraphPad Software,
La Jolla, CA, USA). All the experiment results in this research were repeated at least three times.
p < 0.05 was recognized as the statistical significance between categories.
3. Results and Discussion
3.1. FNDs Preparation
As shown in Figure 1a, the FNDs aggregated in PBS, which made FNDs unable to suspendhomogenously in the culture medium. Therefore, FNDs were conjugated with HSA by sonicating the
FNDs in distilled deionized water for 15 min. They were then mixed with the protein at a weightratio of FND:HSA ≈ 1:1 by gentle shaking at room temperature for 2 h to allow physical adsorption.
After removal of unbound HSA by centrifugation (15,000 rpm for 5 min), the precipitate was thenwashed with phosphate-buffered saline (PBS) [34]. As shown in Figure 1a, FNDs conjugated with HSA
would prevent FNDs aggregation in PBS without interference of fluorescent spectra of FNDs (Figure 1b).
FNDs, like other nanoparticles, are prone to precipitation in physiologic conditions, which makes themdifficult to label the cells homogenously. As described in the Materials and Methods section, FNDs
with carboxylated/oxidized surface functionalities under strong oxidative-acid treatment could havenon-covalent interactions with serum albumin, a colloid-stabilization agent, to prevent agglomerationof FNDs. The FNDs were conjugated with serum albumin without desorption, as reported [36,37].
To test whether the HSA has been taken inside the hASCs, the protein lysates of hASCs after incubation
with HSA alone or FND-conjugated HSA for 4 and 6 h were examined with SDS-PAGE and stainedwith Coomasie blue. The increase of HSA in the cell lysates of hASCs incubated with FND-conjugated
HSA was noted, compared to hASCs incubated with HSA alone or without HSA (Figure S4). Taken
together, these results show that conjugation of FNDs and HSA was stable and could be used forcell labeling.

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Figure 1. (a) Dynamic light scattering measurements of the size distributions of FNDs with or without
Figure 1. (a) Dynamic light scattering measurements of the size distributions of FNDs with or without
HSA conjugation in distilled deionized water and PBS. (b) Fluorescence spectra of FNDs before andHSA conjugation in distilled deionized water and PBS. (b) Fluorescence spectra of FNDs before andafter conjugation with HSA (FND@HSA). The emission spectrum was acquired by laser excitation atafter conjugation with HSA (FND@HSA). The emission spectrum was acquired by laser excitation at
532 nm.
532 nm.

3.2. Characterization of FND—Labeled hASCs
3.2. Characterization of FND—Labeled hASCs
To investigate the number of FNDs that could be loaded into the hASCs, the hASCs were incubated
To investigate
the numberof FNDs
that couldAs
beshown
loadedininto
the 2a,hASCs,
werewith FNDs
at the indicatedtimes andconcentrations.
Figuremanythered hASCs
fluorescent
incubated
the indicatedandconcentrations.
Asµg/mL
shownFNDs
in Figure2a,many redsignals
werewith
notedFNDs
in theatcytoplasm
whentimes
hASCs
were
labeled with 100
for 4 hincubation.
fluorescentsignals
were
noted
in
the
cytoplasm
when
hASCs
were
labeled
with
100
μg/mL
FNDs atfor
The fluorescence of FNDs was taken by the excitation at 532 nm and collection of the emission
4hincubation.
The
fluorescence
of
FNDs
was
taken
by
the
excitation
at
532
nm
and
collectionofthe
685 nm, corresponding to the phonon sidebands of an electronic transition of NV− centers. FNDs wereemissiontaken
at 685into
nm,thecorresponding
toreceptor-mediatedthe phonon sidebandsof an electronictransitionNV- centers.
probably
cytoplasm via
endocytosis,
emitting
a distinctoffluorescence
FNDswaswere
probablytaken
the cytoplasm
via receptor-mediatedendocytosis,
a distinct
which
detectedwiththeintomicroscope,
as previously
reported [32,33]
to account emitting
for the engulfing
fluorescence
which
was
with
the microscope,
as previously
to account
for
FNDs,
and which
could
bedetected
a possible
hypothesisof the hASCs
engulfing reported
the FNDs.[32,33]
We further
appliedtheengulfing
FNDs,
and
which
could
be
a
possible
hypothesis
of
the
hASCs
engulfing
the
FNDs.
We
the background-free detection method to quantify the amount of FNDs taken up by cells. hASCs
further
applied withthe background-freedetection
method to ofquantify
the amount
taken
up by
were
incubated
FNDs at the particle
concentration
5–200 µg/mL,
and oftheFNDs
cellularuptakecells.
hASCs wereanalyzed
incubatedby
with
FNDs at theMMF
particle
concentration
ofremoval
5–200 μg/mL,
the cellularwas
subsequently
a home-built
spectrometer
afterof freeand
FNDs
in theuptakewassubsequently
analyzedbyahome-built
MMF
spectrometer
after
removalof◦medium [34] (Figure 2b). When the hASCs were incubated with 100 µg/mL FNDs at 37 Cfree
for FNDs
four
in the an
medium[34](Figure2b). When
were incubatedwithcell
100wereμg/mL
FNDs
37 °C infor
hours,
averageweightof 49.3
pg (or the~2.7hASCs× 104 particles)
FNDs pernoted.
Asatshown
4
four hours,
average of
weight49.3 pg
× 10inparticles)
FNDs
per cell
werethenoted.
As shown
Figure2c, theanamounts
FNDsoftakenup(orby ~2.7
hASCs
the six time
pointsduring
30 min-to-5hin
Figure2c,theamounts
of
FNDs
taken
up
by
hASCs
in
the
six
time
pointsduringthe30
min-to-5htime frame could be determined by MMF, as described in the Materials and Methods section.
time frame could be determined by MMF, as described in the Materials and Methods section.

Polymers 2019, 11, 1391
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(a)

(b)

(c)

Figure 2.
Characterization ofof fluorescentfluorescent nanodiamond
nanodiamond (FND)-labeled(FND)-labeled human
Figure2. Characterization
human adipose-derived
adipose-derived
stem/stromal
cells(hASCs).
(a)
hASCs
were
incubated
with
100
µg/mL
for
4
h.
The fluorescent
stem/stromal cells (hASCs). (a) hASCs were incubated with 100 μg/mL for 4 h. The fluorescentimagesimagesof
FND-labeled
hASCs
were
obtainedbyafluorescent
microscope.
Cell
nucleistainedbyof FND-labeled hASCs were obtained by a fluorescent microscope. Cell nuclei were were
stainedby 4′,60 ,6-diamidino-2-phenylindole (DAPI). The images were shown: (1) bright-field, (2) DAPI, (3) FNDs,
4
diamidino-2-phenylindole (DAPI). The images were shown: (1) bright-field, (2) DAPI, (3) FNDs, (4)(4) themergedimage,(5) hASCs
without
FNDs.
20 (b)µm.hASCs(b) hASCs
incubatedthemergedimage,
andand(5) hASCs
without
FNDs.
ScaleScalebar: bar:
20 μm.
were wereincubated
with
with
FNDs
at
concentrationsof
10–200
µg/mL
for
four
hours.
Quantification
of
FNDs
in
the
cells
was
FNDs at concentrations of 10–200 μg/mL for four hours. Quantification of FNDs in the cells was
performed
andanalyzedbymagneticallymodulated
fluorescence(MMF).
(c)
hASCs
were
incubated
performed and analyzed by magnetically modulated fluorescence (MMF). (c) hASCs were incubated
with
in a
manner (half(half toto five
five hours)
hours) at
at 100100 μg/mL.
µg/mL. The
FNDs were
werewith FNDs
FNDs ina time-course
time-course manner
The intracellular
intracellular FNDs
subsequently
analyzedby
MMF.
subsequently analyzed by MMF.

3.3. Proliferations and Differentiation of hASCs Not Affected by FNDs
3.3. Proliferations and Differentiation of hASCs Not Affected by FNDs
The intensity of fluorescent signals of FNDs increased in a dose-dependent manner, and the
The intensity of fluorescent signals of FNDs increased in a dose-dependent manner, and thesignals of FNDs in cells could still be detected with increasing passages of cells (Figure 3a). Since FNDs
signals of FNDs in cells could still be detected with increasing passages of cells (Figure 3a). Since
have been illustrated as cell-tracking materials [32,38,39], we next examined whether the FNDs showed
FNDs have been illustrated as cell-tracking materials [32,38,39], we next examined whether
the FNDs
cytotoxicity in hASCs. MTT analysis, a cell viability assay, was performed with 1 × 105 hASCs labeled
showed cytotoxicity in hASCs. MTT analysis, a cell viability assay, was performed with 1 × 105 hASCs
with FNDs in a dose-dependent manner with one- or five-day incubation. No significant differencelabeled with FNDs in a dose-dependent manner with one- or five-day incubation. No significant
was noted in each group in terms of cytotoxicity and cell proliferation (Figure 3b). Flow cytometry
difference was noted in each group in terms of cytotoxicity and cell proliferation (Figure 3b). Flowanalyses were performed to compare the expression pattern of stem/stromal cell-associated specificcytometry analyses were performed to compare the expression pattern of stem/stromal cellsurface markers of hASCs with or without FND labeling. Positive expression of CD29, CD44, CD90,
associated specific surface markers of hASCs with or without FND labeling. Positive expression of
and CD105 were observed in the hASCs regardless of the presence of FND, but was negative for CD45
CD29, CD44, CD90, and CD105 were observed in the hASCs regardless of the presence of FND, but(Figure 3c). It was illustrated that FND-labeled hASCs possessed consistent characteristic mesenchymal
was negative for CD45 (Figure 3c). It was illustrated that FND-labeled hASCs possessed consistentstem/stromal cell (MSC) phenotypes as hASCs.
characteristic mesenchymal stem/stromal cell (MSC) phenotypes as hASCs.
To further evaluate the effect of FNDs on hASCs’ multilineage differentiation capability of
To further evaluate the effect of FNDs on hASCs’ multilineage differentiation capability of
hASCs, cells were pre-incubated with 100 µg/mL FNDs and cultured in an adipogenesis, osteogenesis,
hASCs, cells were pre-incubated with 100 μg/mL FNDs and cultured in an adipogenesis,or chondrogenesis induction medium, respectively, for three weeks to allow cell differentiation.
osteogenesis, or chondrogenesis induction medium, respectively, for three weeks to allow cell
As shown in the Supplementary Figure S1a, FND-labeled hASCs displayed no difference in adipogenic
differentiation. As shown in the Supplementary Figure S1a, FND-labeled hASCs displayed nodifferentiation compared to the control sample, as evaluated by Oil-Red O staining. The hASCs withdifference in adipogenic differentiation compared to the control sample, as evaluated by Oil-Red O
FND labeling also showed no differences in osteogenesis compared to the control sample, as determined
staining. The hASCs with FND labeling also showed no differences in osteogenesis compared to theby Alizarin Red S staining (Supplement Figure S1b). Similar results were found in chondrogenic
control sample, as determined by Alizarin Red S staining (Supplement Figure S1b). Similar results
differentiation stained with Alcian Blue, which indicates that the FND-labeled hASCs maintained
were found in chondrogenic differentiation stained with Alcian Blue, which indicates that the FNDlabeled hASCs maintained chondrogenic differentiation activity (Supplement Figure S1c). Overall,Polymers 2019, 11, 1391
Polymers 2019, 11, x FOR PEER REVIEW

9 of 16
9 of 16chondrogenic
differentiation
activity (Supplement
FigureS1c). Overall,the FND-labeled
hASCs
the FND-labeled
hASCs maintained
their potential
for adipogenic,
osteogenic,
and chondrogenic
maintained
their potential for adipogenic, osteogenic, and chondrogenic differentiation.
differentiation.

(a)

(b)

(c)

Figure3. 3. (a)Fluorescence intensity
intensityofofFND-labeled
FND-labeled
hASCs
detectedpassage.
Figure(a) FluorescencehASCs
waswasdetected
afterafter
each eachpassage.
1 × 105
5
1 ×hASCs
10 hASCs
labeled50, 100,
150 µg/mL).
Valuesmeans± S.E.M.
were werelabeledwith with
FNDsFNDs(0, 50,(0,100,
150 μg/mL).
Valuesare aremeans± S.E.M.
fromfrom
three
three
independent
experiments.
Data
were
assessedusing
one-way
ANOVA,
followedby
Turkey’s
independent experiments. Data were assessed using one-way ANOVA, followed by Turkey’s
5
multiplecomparison
tests.
(b)(b)
1 ×1 10
were
with
FNDs
(0,(0,
25,25,
50,50,
100µg/mL)
forforone
5 hASCs
weretreatedtreated
with
FNDs
100
μg/mL)
one
multiplecomparison
tests.
× 10hASCs
and
five
days,
followedby
MTT
analysis.
Valuesrepresent
the
means
±
S.D.
of
three
independentand five days, followed by MTT analysis. Values represent the means ± S.D. of three independent
experiments.
Data
were
assessedusing
T tests.
(c)(c)
Surfacemarkers
of of
hASCs.
Representativeflow
experiments.
Data
were
assessedusing
T tests.
Surfacemarkers
hASCs.
Representativeflow
cytometry
analysisdemonstrated
positivesformesenchymal
stem/stromalcells
markers
CD29,
CD44,
cytometry analysis demonstrated positives for mesenchymal stem/stromal cells markers CD29, CD44,
CD90,
and
CD105,
butbutnegative
forfor
CD45.
Blue
lines
indicatethetheisotypecontrol.
FITC-A,fluorescein
CD90,
and
CD105,
negative
CD45.
Blue
lines
indicateisotypecontrol.
FITC-A,fluorescein
isothiocyanate
area.
isothiocyanate area.

3.4. hASCs Maintained the Volume of Xenografts Containing Decellularized Cartilage and Fibrin Glue Polymer
3.4. hASCs Maintained the Volume of Xenografts Containing Decellularized Cartilage and Fibrin Glue
Three groups of xenografts were performed in an in vivo experiment, including ECM-glue,
Polymer
ECM-hASC-glue, and hASC-glue, as described in the Materials and Methods section. The combination
Three groups of xenografts were performed in an in vivo experiment, including ECM-glue,of fibrin glue polymer and diced decellularized cartilage ECM served as an appropriate 3D scaffold
ECM-hASC-glue, and hASC-glue, as described in the Materials and Methods section. The
with adhesive properties maintaining the xenograft constructs [23]. Furthermore, fibrin is applied
combination of fibrin glue polymer and diced decellularized cartilage ECM served as an appropriate
extensively for clinical applications because of its clinical safety [27]. To test whether the fibrin
3D scaffold with adhesive properties maintaining the xenograft constructs [23]. Furthermore, fibrin
scaffolds containing diced decellularized cartilage ECM could make in vivo chondrogenesis of hASCs
is applied extensively for clinical applications because of its clinical safety [27]. To test whether the
without in vitro induction, xenografts were implanted into the subcutaneous layer of nude mice forfibrin scaffolds containing diced decellularized cartilage ECM could make in vivo chondrogenesis ofthree months (Figure 4a above). The diameter of xenografts post three month-implantation from thehASCs without in vitro induction, xenografts were implanted into the subcutaneous layer of nude
ECM-hASC-glue group was approximately 12 mm (Figure 4a, bottom right), whereas the diameter
mice for three months (Figure 4a above). The diameter of xenografts post three month-implantationof xenografts post three-month implantation from the ECM-glue group was approximately 6 mmfrom the ECM-hASC-glue group was approximately 12 mm (Figure 4a, bottom right), whereas thediameter of xenografts post three-month implantation from the ECM-glue group was approximately

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(Figure 4a bottom left). Three months afterwards, the volume of the xenografts from the ECM-glue
and ECM-hASC-glue groups were 64.09% ± 15.8 and 98.97% ± 17.35 compared to the original volumeof xenografts, respectively (Figure 4b). As shown in Supplementary Figure S2, the appearance on
the skin of the hASC-glue group was flat, as indicated by the arrows one month after implantation.
To further examine whether the hASCs were associated with maintenance of the volume of xenografts,
histological analysis of the xenografts was performed. As shown in Figure 4c,d, the increased numberof cell nuclei stained by DAPI (blue dots) was noted in the group of ECM-hASC-glue compared to
ECM-glue, indicating that hASCs could significantly maintain the xenograft volume in vivo.
The combination of the diced irradiated cartilages and fibrin glues has been used in the clinicaltreatment in recent years. One of the disadvantages of the current method is the reabsorption of the graftwith decreased volume, which made the clinical outcome unsatisfactory. Therefore, stem-cell-based
therapies combined with scaffolds could be one of the promising strategies in chondrogenesis.
The grounded ECM is a loose, pulverized material; hence, the xenograft constructs made of ECM-alone
and ECM-hASCs could not be maintained without fibrin glue polymer in this study. Fibrin glue
polymers used in this study could be maintained for 5 days, which might contribute to the formationof the xenograft construct in our experiment [28]. If we include glue-alone or hASCs-alone in theexperimental group in our three-month experimental design, the volume of the xenografts could
not be maintained for three months. The xenograft constructs of hASC-glue which disappeared one
month after implantation might tell us the importance of ECM in volume maintenance. Taken together,
the xenograft constructs could be supported by the decellularized ECM, and the volume of the xenograft
could be maintained by hASCs. These findings are important for clinical applications, as they provide
valuable
information
for understanding
the mechanism of supportive volume in cartilage repair.
Polymers 2019,11, x FOR PEER
REVIEW
11 of 16

(a)

(b)

(c)

(d)

(e)

Figure 4. Cont.

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Figure 4.
4. Thexenografts composed
diced decellularized
decellularized cartilage
fibrin glue
glue polymer
polymer
FigureThe xenografts
composed ofof dicedcartilage ECM
ECM andand fibrin
with oror without
without FND-labeled
FND-labeled hASCs
hASCs were
were transplanted
transplanted subcutaneously
subcutaneously into
nude mice
mice forfor three
three
with
into nudemonths. (a)ECM
andfibringlue
polymerwithormonths.
(a)Macroscopic
Macroscopicappearance
appearanceofofxenografts
xenograftscontaining
containing
ECM
andfibringlue
polymer
withwithout
FND-labeled
hASCs
beforeimplantation(above)andthreemonthsafterwards(below).
(b)
or without FND-labeled hASCs before implantation (above) and three months afterwards (below).
Volumeof thexenografts
weremeasured.
There
waswasa statistic
significant
differencein thevolumeof(b)Volumeof thexenografts
weremeasured.
Therea statistic
significant
differencein thevolumexenograft
betweenthethegroupsof ECM-glue
andandECM-hASC-glue.
The Thevalueswereweremeans± S.D.
=
ofxenograft
betweengroupsof ECM-glue
ECM-hASC-glue.
values
means
± (n
S.D.
8).=**8).
p <**0.01
control.
DataData
werewere
assessedusing
T tests
for foranalysis.
(c) Histologicalanalysisof(np < versus
0.01 versus
control.
assessedusing
T tests
analysis.
(c) Histologicalanalysis
xenografts
in in
both
groups.
ofofcellofxenografts
bothgroups.Comparison
Comparison
celldensity
densityby
byDAPIDAPIbetween
betweengroups
groups ofof ECM-glue andECM-hASC-glue three months afterwards
afterwards (Original(Original magnification
magnification ×× 400). (d)Cellnumbers were(d)quantified under microscope perper field,
field, asas described
describedin
inthe
theMaterials
Materialsand
andMethods
Methodssection,section,by
byImage
ImageJ
ECM-hASC-gluegroup
has hassignificantly
increased
cell numbers
comparedto the to
ECMJsoftware.
software.The
The
ECM-hASC-gluegroupsignificantly
increased
cell numbers
compared
theglue group.group.
The values
were mean
S.E.M.
(n = 8).(n**=p 8).
< 0.01* p < 0.05* pversus
ECM-glue
The values
were ±mean± S.E.M.
** pversus< 0.01 control.
versus control.
< 0.05control.
versusData wereassessedusing T tests
forT analysis.
(e) Identificationof FND-labeled
hASCs in hASCs
a xenograftcontrol.
Data
were assessedusing
tests for analysis.
(e) Identificationof FND-labeled
in axenograft tissue section by confocal microscopy: (1) H&E staining image, (2) confocal fluorescent image
without time gating, (3) time-gated confocal fluorescent image, and (4) merged image of (1) and (3).
Black arrows denote FND-labeled hASCs. Scale bar: 50 µm. (f) Quantitative detection of FND-labeled
hASCs with MMF. The corresponding percentages of FND-labeled hASCs found in the implanted
xenografts 90 days later. The values were means ± S.E.M. (n = 3). Data were assessed using T tests
for analysis.

3.5. Long-Term Detection of FND-Labeled hASCs with an In Vivo Chondrogenesis Model
To further identify whether the implanted FND-labeled hASCs could be detected in the xenografts,
we tracked the hASCs using the unique property of FNDs. FNDs contain the fluorophores NV− centerswith a fluorescence lifetime (τ) longer than 15 ns, which is substantially longer than that (τ ≈ 1–4 ns)of the endogenous and exogenous fluorophores commonly used in cell biology [40]. Deparaffinizedsamples with H&E staining were used for FNDs imaging using time-gated confocal fluorescencemicroscopy to achieve background-free detection [34]. As shown in Figure 4e, several cell nuclei
were noted around the surface of decellularized ECM (Figure 4e(1)). The signals of FND-labeled
hASCs were interfered by the strong background fluorescence and autofluorescence under a confocal
microscope (Figure 4e(2)). The signals of FNDs could be clearly detected using time-gated confocal
fluorescence microscopy (Figure 4e(3)). As shown in Figure 4e(4), the merged images clearly showed
the location of FND-labeled hASCs in the H&E staining. Taken together, these findings indicated that
FND-labeled hASCs could be detected in the xenograft without interference of background fluorescence
three months afterwards. As shown in Figure 4f, numbers of implanted FND-labeled hASCs in the
xenografts can be quantified by MMF, as described in the Materials and Methods section. To ourknowledge, our study is probably the first experiment to detect FND-labeled cells for three months in
an animal model.

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On the strength of the chemical robustness of the nanomaterial, the unique magneto-opticalproperties
of FNDs
still preserved
and(1)
theirsignals
could bereadily recoveredfromtissuesectionwere
by confocal
microscopy:
H&Efluorescencestaining image,(2) confocal
fluorescentimage without
the acidtime
digests
by magnetic
modulation
at the concentrationas 1 image
ug/mL.
recoveryrate,gating,(3) time-gatedconfocal fluorescentimage, and as(4)low
mergedof The(1) and(3). Black
determinedby spiking
a known amountof Scale
FNDsbar:into50themouse
tissue followedby digestion
througharrowsdenote FND-labeled
hASCs.
μm.
(f) Quantitative
detectionof FND-labeled
the samehASCs
process,more
90% [34]. Suchquantitative
analysis hASCs
is inaccessible
molecular
withwasMMF.
Thethancorresponding
percentagesof FND-labeled
found inwith
the implanted
fluorophores
such 90
as daysorganic
dyes
fluorescentproteins
becausethewerelackassessedof chemical
xenografts
later.
The andvalueswere means± S.E.M.
(n = 3). of
Data
usingstabilities
T tests forin
analysis.
strong acids.
3.6.3.6.
Functional
In Vivo
Chondrogenesis
Functional
In Vivo
Chondrogenesis

Allspecimens
specimenswere
were sent
sent for histologicalBlue
andandMasson’sAllhistological analysis
analysis andandtreatedtreatedwith
withAlcian
AlcianBlue
Masson’s
Thrichrome
staining
fordetectionofglycosaminoglycans
andcollagen,respectively.
As
shown
in in
Thrichrome staining for detection of glycosaminoglycans and collagen, respectively. As shown
Figure5a, 5a,AlcianBluestaining
showedincreased
blue
staining
andandredreddots(cell(cellnuclei)in theFigureAlcianBluestaining
showedincreased
blue
staining
dots
nuclei)in theinterstitial
space
in ECM-hASC-glue
xenografts
comparedto the
ECM-glue
xenografts.
Increasedinterstitial
space
in ECM-hASC-glue
xenografts
comparedto the
ECM-glue
xenografts.
Increasedcollagen(blue
color)
waswasalsoalso
notedin theMasson’s
Trichrome
staining.
Moreover,thetheresultswithcollagen(blue
color)
notedin theMasson’s
Trichrome
staining.
Moreover,results
with
AlcianBlueandandNuclearFastFast
RedRed(red(redcolor)
staining
showedhighercellularity
in ECM-hASC-glue
AlcianBlue
Nuclearcolor)
staining
showedhighercellularity
in ECM-hASC-gluegroup
comparedto thegroup.group.
This resultcompatible
with ourwith
previous
results, asresults,
shown asgroup
compared
toECM-glue
the ECM-glue
This isresult
is compatible
our previous
in Figure
These4c.
findings
indicatedindicatedthat increased
glycosaminoglycans
and collagenmight mightbeshown 4c.
in Figure
These findingsthat increased
glycosaminoglycans
and collagensecretedby thebychondrocyte-likecells derived
from fromthe hASCs.
To further
validate
whetherthethebe secreted
the chondrocyte-likecells derived
the hASCs.
To further
validate
whether
FND-labeled
hASCs
underwentchondrogenic
differentiation,thetheimmunofluorescencestainings
of of
FND-labeled
hASCs
underwentchondrogenic
differentiation,
immunofluorescencestainings
thethexenografts
wereperformed
for foraggrecanandandcollagenII withthe theconfocalmicroscope.
As As
shown
xenografts
wereperformed
aggrecancollagenII withconfocalmicroscope.
shown
in Figure5b,5b,
colocalization(redcolor)color)andand
aggrecan(greencolor)
in FigurecolocalizationofofFNDs-labeled
FNDs-labeled hASCs (redaggrecan(greencolor)
waswasnoted.
noted.
Similarresults
were
noted
in
collagenII
staining
inFigure5c.
Thecolocalizationofsignals
Similar results were noted in collagen II staining in Figure 5c. The colocalization of signals of FNDs
of FNDsand chondrocyte-markers,
includingaggrecancollagetype
type II,
II, might
vivo
and chondrocyte-markers,
includingaggrecanandandcollagemight indicate
indicatein invivochondrogenesis
ofhASCs.
Taken
together,functionalinvivo
chondrogenesisofthe
FND-labeledchondrogenesis of hASCs. Taken together, functional in vivo chondrogenesis of the FND-labeled
hASCs
in xenografts
waswasfound,
andandthese
xenografts
containdecellularized
ECM
andandfibringlue
hASCs
in xenografts
found,
these
xenografts
containdecellularized
ECM
fibringlue
polymerthreemonthsafterwards.
polymerthreemonthsafterwards.

(a)Figure 5. Cont.

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13 of 16(b)(c)

Figure 5. Functional in vivo chondrogenesis of hASCs. (a) The presence of glycosaminoglycans
Figure5. Functionalin vivo chondrogenic
chondrogenesis
of hASCs. (a)byThepresenceglycosaminoglycans
andandcollagendemonstrated
differentiation
AlcianBlueofand
Masson’s Thrichromecollagendemonstratedchondrogenic
differentiationbyAlcianBlue
and
Masson’s
Thrichrome
staining, separately. Alcian Blue, Nuclear Fast Red, and Masson’s Thrichrome staining of the xenografts
staining,
separately. Alcian
Blue, Nuclear
Fastthree
Red,months
and Masson’s
Thrichrome
stainingof thefrom
ECM-hASCs-glue
and ECM-gluegroupsafterwards.
Upper panel
represents
xenografts
from
ECM-hASCs-glue
ECM-glue
three months
afterwards.
Upper
panel
the
ECM-gluegroup.
Lower paneland
represents
the groups
ECM-hASCs-gluegroup.
Masson’s
Trichrome
represents
theECM-gluegroup.
Lower
panel
representstheECM-hASCs-gluegroup.
Masson’s
staining of the ECM-hASCs-glue and ECM-glue groups after three months implantation (Original
Trichrome stainingof the
ECM-hASCs-glue
and ECM-gluegroupsafter three months implantation
magnification
× 200).
(Arrows
indicate the interstitialspaces.)(b) Immunofluorescent
staining for(Original
magnification
×200).
(Arrows
indicatetheinterstitialspaces.)(b) inImmunofluorescent
Aggrecan for identifying glycosaminoglycan (green) and FND-labeled hASCs (red)ECM-hASCs-glue
staining
for Aggrecan
identifying
glycosaminoglycan(green)and FND-labeled
hASCs(red) inin
and
ECM-gluegroups.for(c)Fluorescenceimaging of type
II collagen(green) were
presented
ECM-hASCs-glue andand ECM-glue
ECM-gluegroups.
groups.Scale(c) Fluorescence
ECM-hASCs-gluebar: 50 µm. imaging of type II collagen (green) were
presented in ECM-hASCs-glue and ECM-glue groups. Scale bar: 50 μm.

4. Conclusions

4. Conclusions
This study demonstrates that the volume of xenograft constructs could be better maintained withincreased
cellularity
in the ECM-hASCs-gluegroup
comparedto the ECM-glue
addition,
This study
demonstrates
that the volumeof xenograft
constructs
could begroup.
better In
maintained
increased
cartilage
proteins
were
noted
in
the
ECM-hASCs-gluegroup
byMasson’strichromewith increased cellularity in the ECM-hASCs-glue group compared to the ECM-glue group.andIn
Alcianblue
staining.cartilage
Signalsproteinsof chondrogenic
markers,
as aggrecan andcollagentype IIaddition,increased
were notedin the such
ECM-hASCs-gluegroup
by Masson’s
colocalized
withAlcian
FND signals,
might indicate
in chondrogenic
vivo chondrogenesis
of hASCs
xenografts
containing
trichrome andblue staining.
Signals ofmarkers,
such asinaggrecan
andcollagendecellularized
cartilage
ECM
andfibringlue
polymers.
Furthermore,
weintroduceda
FND-based
type II colocalized with FND signals, might indicate in vivo chondrogenesis of hASCs in xenografts

containing decellularized cartilage ECM and fibrin glue polymers. Furthermore, we introduced a
FND-based method combining cell-detecting technology with quantitative tracking of transplanted
cells in vivo, which may warrant future stem/stromal cell research into translational research.

Polymers 2019, 11, 1391

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method combining cell-detecting technology with quantitative tracking of transplanted cells in vivo,
which may warrant future stem/stromal cell research into translational research.
Supplementary Materials: The following are available online at http://www.mdpi.com/2073-4360/11/9/1391/s1,
Figure S1: FNDs result in no alteration on cell differentiation derived from hASCs. Figure S2: Appearance of
hASC-glue group nude mice before (left) or after one-month (right) implantation. Figure S3: ECM preparation.
Author Contributions: Conceptualization, Y.-C.W. (Yi-Chia Wu) and M.-L.H.; methodology, H.-H.L. and L.-J.S.;
validation, L.-J.S.; formal analysis, Y.-R.K.; data curation, H.-M.D.W.; writing—original draft Y.-C.W. (Yi-Chia Wu)
and Y.-C.W. (Ya-Chin Wang); writing—review & editing, W.-T.W. and H.-H.L., Supervision, C.-S.L. and J.Y.
Funding: This research was supported by the Southern Taiwan Science Park Bureau, South Taiwan Smart
Manufacturing Industrial Clusters, Taiwan under contract 106SMIC-MC06. This study is partially supported by
Kaohsiung Medical University Research Center Grant (KMU-TC108A02); grants from Academia Sinica, Taiwan to
Yi-Chia Wu (AS-TM-108-02-01). This study was partially funded by grants from CGMH at Linko of Taiwan toJohn Yu (OMRPG3C0041 to OMRPG3C0044); Ministry of Science and Technology, Taiwan to Yi-Chia Wu (MOST
103-2628-B-037-002-MY3); Yi-Chia Wu (kmtth-105-011; kmtth104-011); grants from Kaohsiung Medical University
Hospital to Yi-Chia Wu (kmuh98-8G42 and kmuh99-9M54).
Acknowledgments: We would like to express our gratitude to the Center for Research Resources and Development(CCRD) of Kaohsiung Medical University for the technical assistance. We thank Huan-Cheng Chang for technicalsupport of the MMF. We thank Yen-Yiu Liu for constructive criticism of the manuscript.
Conflicts of Interest: The authors declare no conflict of interest.

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