Abstract

   Heterogeneity of mesenchymal stem cells (MSCs) influences the cell
   therapy outcome and the application in tissue engineering. Also, the
   application of subpopulations of MSCs in cartilage regeneration remains
   poorly characterized. CD146+ MSCs are identified as the natural
   ancestors of MSCs and the expression of CD146 are indicative of greater
   pluripotency and self-renewal potential. Here, we sorted a CD146^+
   subpopulation from adipose-derived mesenchymal stem cells (ADSCs) for
   cartilage regeneration.

   Methods: CD146^+ ADSCs were sorted using magnetic activated cell
   sorting (MACS). Cell surface markers, viability, apoptosis and
   proliferation were evaluated in vitro. The molecular signatures were
   analyzed by mRNA and protein expression profiling. By intra-articular
   injections of cells in a rat osteochondral defect model, we assessed
   the role of the specific subpopulation in cartilage microenvironment.
   Finally, CD146^+ ADSCs were combined with articular cartilage
   extracellular matrix (ACECM) scaffold for long term (3, 6 months)
   cartilage repair.

   Results: The enriched CD146^+ ADSCs showed a high expression of stem
   cell and pericyte markers, good viability, and immune characteristics
   to avoid allogeneic rejection. Gene and protein expression profiles
   revealed that the CD146^+ ADSCs had different cellular functions
   especially in regulation inflammation. In a rat model, CD146^+ ADSCs
   showed a better inflammation-modulating property in the early stage of
   intra-articular injections. Importantly, CD146^+ ADSCs exhibited good
   biocompatibility with the ACECM scaffold and the CD146^+ cell-scaffold
   composites produced less subcutaneous inflammation. The combination of
   CD146^+ ADSCs with ACECM scaffold can promote better cartilage
   regeneration in the long term.

   Conclusion: Our data elucidated the function of the CD146^+ ADSC
   subpopulation, established their role in promoting cartilage repair,
   and highlighted the significance of cell subpopulations as a novel
   therapeutic for cartilage regeneration.

   Keywords: CD146, cartilage tissue engineering, mesenchymal stem cell,
   scaffold, extracellular matrix

Introduction

   Articular cartilage, lacking blood vessels, lymphatic vessels, and
   nerves, is not subjected to systemic regulation [69]^1. Cartilage is
   susceptible to damage, and acute lesions without proper treatment may
   result in post-traumatic osteoarthritic progression [70]^2^, [71]^3.
   Traditionally, bone marrow stimulating procedures, including abrasion,
   subchondral drilling, and microfracture techniques, can mobilize
   progenitor cells from the subchondral bone to promote cartilage repair
   [72]^4^-[73]^6. However, hyaline repair tissue cannot be consistently
   induced, and most often it does not suffice to fill the entirety of the
   defect. Currently, autologous chondrocyte implantation is used in
   clinical practice [74]^7, but issues of limited availability,
   de-differentiation, and functional loss during in vitro culturing limit
   its application [75]^8. Stem cells, because of their high cell
   viability, and multilineage differentiation capacity, are therefore
   being investigated as alternative cell sources for cartilage
   regeneration [76]^9^-[77]^12.

   Research on stem cell-based tissue engineering has until now mostly
   concentrated on harvesting mesenchymal stem cells (MSCs) from different
   sources, such as bone marrow [78]^13^, [79]^14, adipose tissue [80]^15,
   synovium [81]^16, the bloodstream [82]^17, umbilical cord/Wharton's
   jelly [83]^18, amniotic fluid [84]^19 and the amnion [85]^20, for
   exploring their self-renewal and differentiation capacities and their
   application in in vivo cartilage repair. However, MSCs are a
   heterogeneous population, which cannot be identified and isolated using
   single surface markers [86]^21^, [87]^22, and therefore MSC-based
   clinical trial outcomes vary [88]^21. Hence, the cellular heterogeneity
   of MSCs affects the treatment outcome. In this context, the use of
   specific subpopulations of MSCs in tissue regeneration is an emerging
   idea and represents an innovative approach.

   CD146, also known as MUC18, MCAM, Mel-CAM, or S-Endo1, is a
   transmembrane glycoprotein and an adhesion molecule of the
   immunoglobulin (Ig) superfamily [89]^23^, [90]^24. CD146 was initially
   known as an endothelial biomarker and a marker for melanoma progression
   [91]^25. Later, it was also recognized as a marker for pericytes
   [92]^24^, [93]^26, which have been identified as the natural ancestors
   of MSCs [94]^27^, [95]^28. In human tissues, CD146^+ cell was regarded
   as the mural cells of blood micro vessels [96]^29^, [97]^30. Previous
   reports also showed that CD146 was associated with cell migration and
   self-renewal [98]^31^, [99]^32. Hagmann et al. found that CD146^+ bone
   marrow MSCs (BMSCs) increased GAG/DNA content after enrichment
   [100]^33. Wu et al. reported that a CD146^+ subpopulation from human
   umbilical cord cells could treat arthritis and provide an
   anti-inflammatory protective microenvironment by suppressing IL-6
   [101]^34. Su et al. showed that CD146^+ chondroprogenitors expressed
   higher levels of an MSC-specific marker and had better chondrogenic
   differentiation capacity [102]^35. Thus, we hypothesized that CD146^+
   subpopulations have the potential to promote better cartilage repair.

   For clinical applications, sorting CD146^+ cells from adipose-derived
   MSCs (ADSCs) has many advantages. ADSCs are maintainable in vitro for
   longer times than BMSCs and exhibit stable population doubling with
   higher proliferation and lower senescence rates. Fewer ADSC passages
   are required to obtain adequate cell numbers. hADSCs can be derived
   from lipoaspirate, which can be harvested through tumescent abdominal
   liposuction techniques. A sufficient number of cells can be obtained in
   one attempt which avoids the morbidity associated with harvest
   [103]^10^, [104]^36. A recent study compared the differences of ADSCs
   and BMSCs at single- and bulk-cell levels on the treatment of
   osteoarthritis, they found ADSCs as a more stable and controllable stem
   cell source, was more adaptable to surviving in the hypoxic articular
   cavity niche, and exhibited superiority in regulating inflammation
   [105]^37.

   In this study, we report a CD146^+ MSC subpopulation-based cartilage
   tissue engineering strategy. The CD146^+ subpopulation was enriched
   from human ADSCs. Cellular characteristics, including the expression of
   the surface marker, immune character, viability, proliferation gene and
   cytokine expression patterns were identified in vitro. Further, the
   role of the CD146^+ subpopulation in the cartilage microenvironment was
   explored in vivo. To provide a more suitable microenvironment for cell
   transplantation, we also developed an articular cartilage extracellular
   matrix (ACECM) cell-scaffold composite and investigated its
   biocompatibility and its role in repairing cartilage defects. We
   successfully identified and isolated CD146^+ subpopulations from
   hADSCs, investigated their cytological functions, and established their
   role in cartilage regeneration.

Methods

hADSC culture

   Human adipose-derived mesenchymal stem cells (hADSCs) at passage 4 were
   provided by the National Cell Bank for Tissue Engineering (Zhejiang,
   China). hADSCs were plated in growth medium consisting of Dulbecco's
   Modified Eagle Medium/ Fisher12 (DMEM/F12) medium (10% fetal bovine
   serum, 1% penicillin-streptomycin added), followed by incubation in a
   humidified incubator at a temperature of 37 °C. Cells were harvested
   when they reached ~80% confluence using 0.25% trypsin in
   ethylenediaminetetraacetic (EDTA; Invitrogen).

Isolation of CD146+ cells from hADSCs

   CD146+ cells were isolated from a suspension of passage 5 hADSCs by
   magnetic activated cell sorting (MACS) using anti-CD146 magnetically
   labeled antibodies (catalog 130-093-596, Miltenyi Biotec) (Figure
   [106]1A).

Figure 1.

   [107]Figure 1
   [108]Open in a new tab

   Overview of experimental design. (A) MACS enrichment of CD146^+ ADSCs.
   (B) Comparison of cytological characteristics between ADSCs and CD146^+
   ADSCs in vitro. CD146^+ ADSCs retained good viability, proliferation
   ability, and stem cell character after sorting. CD146^+ ADSCs also
   showed pericyte characteristics, better immune properties, and
   inflammation regulation ability. (C) A schematic illustration of the
   intra-articular injection of CD146^+ ADSCs in rats. (D) Flow chart of
   the preparation of the cell-scaffold composite for long term cartilage
   repair in rabbits.

Flow cytometry

   A 100-mL aliquot of hADSCs or freshly sorted CD146^+ cells suspension
   (1×10^6/mL) was transferred to the test pipe. Cells were stained with
   anti-human antibodies against the following cell surface markers:
   CD34-PE (BD Pharmingen), CD45-FITC (BD Pharmingen), CD73-PE (BD
   Pharmingen), CD90-FITC (BD Pharmingen), CD105-PE (BD Pharmingen),
   CD146-PE (BD Pharmingen), and HLA-DR-FITC (BD Pharmingen). The cells
   were incubated in the dark for 30 min, collected on a FACS Calibur flow
   cytometer, and the results were analyzed using Cell-Quest for Macintosh
   Software.

Western blotting

   Total proteins were extracted from hADSCs and freshly sorted CD146^+
   cells using RIPA buffer with PMSF. After quantifying the protein
   concentration using a BCA protein assay kit, proteins were subjected to
   SDS-PAGE and transferred to a PVDF membrane. The membrane was incubated
   with primary antibodies against the following proteins: HLA-1 (1:1000;
   Abcam), MHC-II (1:10,000; Abcam), CD40 (1:500; Abcam), CD80 (1:500;
   Abcam), CD86 (1:5000; Abcam), NG2 (1:5000; Novus Biologicals), PDGFR-β
   (1:1000; Abcam), RGS5 (1:1000; Novus Biologicals), and GAPDH
   (1:10,000). Subsequently, the membrane was washed with Tris-buffered
   saline (TBS) containing 0.05% Tween-20 and incubated with an anti-mouse
   or an anti-rabbit HRP-conjugated secondary antibody. The
   antigen-antibody reaction was visualized using enhanced
   chemiluminescence assay (Western Luminescent Detection Kit) and
   documented using Gel Doc (Bio-Rad).

Immunocytochemical and immunofluorescence staining

   For immunocytochemical staining, cells were fixed with 4%
   paraformaldehyde (PFA) for 20 min at room temperature, treated with
   methanol for 10 min, washed with PBS, permeabilized with 0.25% Triton
   X-100 in PBS for 20 min, and blocked with 5% goat serum in 0.25% Triton
   X-100 in PBS for 20 min. Next, cells were incubated with anti-HLA Class
   I (Abcam, ref. ab23755) and anti-MHC-II (Abcam, ref. ab157210) at 4°C
   overnight. The immunodetection was performed using DAB (MXB
   biotechnologies), following the manufacturer's instructions. Cells were
   viewed with an Olympus BX51 light microscope (Olympus, Tokyo, Japan).
   The images were captured using an Olympus CCD DP71 (Olympus).

   For immunofluorescence staining, cells were fixed with 4% PFA for 20
   min at room temperature, treated with methanol for 10 min, washed with
   PBS, permeabilized with 0.25% Triton X-100 in PBS for 20 min, and
   blocked with 5% goat serum in 0.25% Triton X-100 in PBS for 20 min.
   Subsequently, cells were incubated with anti-CD146 (Abcam),
   anti-PDGFR-β (Abcam), anti-RGS5 (Novus Biologicals), or anti-NG2/MCSP
   (Novus Biologicals) at 4°C overnight. On the the next day, cells were
   washed and incubated with secondary antibody in 5% goat serum for 1 h
   at room temperature. Cells were counterstained with a 1:1000 dilution
   of DAPI to label the nuclei and viewed with an Olympus BX51 light
   microscope (Olympus, Tokyo, Japan). The images were captured by using
   an Olympus CCD DP71 (Olympus).

Quantitative real-time PCR

   Total RNA from hADSCs and freshly sorted CD146^+ cells was isolated
   using Trizol following the manufacturer's instructions (Sigma (T9424)).
   The cDNA was synthesized using the SuperScript TM III First-strand
   synthesis system for RT-PCR, cat. No: 18080-051 (Invitrogen).
   Quantitative RT-PCR was performed using the Power SYBRgreen PCR Master
   Mix (ABI, P/N: 4367659). Primers were shown in Table [109]S1. The PCR
   cycling condition included an initial denaturation at 95°C for 2 min,
   40 cycles of 95°C for 15 s, 58°C for 15 s, and 72°C for 35 s, and a
   final extension at 72°C for 5 min. The results were analyzed using
   Bio-Rad iQ5 optical system software.

Cell proliferation evaluation by Cell Counting Kit-8 (CCK-8)

   Cells were seeded at 3000 cells per well in a 96-well plate and
   cultured in a humidified incubator at a temperature of 37°C. The
   relative cell number was determined using CCK8 by OD (absorbance) value
   after 1, 3, and 5 days of culturing, following the manufacturer's
   instructions. The morphology of cells on was observed by phase-contrast
   microscopy (Olympus, BH-2).

Apoptosis and viability analysis

   The hADSCs and freshly sorted CD146^+ cells were trypsinized using
   0.25% trypsin-EDTA and harvested by centrifugation. Apoptosis of ADSCs
   and CD146+ cells was detected using Muse Count & Viability Kit
   (MCH100102). Briefly, cells were incubated with Muse Annexin V & Dead
   Cell reagent for 20 min at room temperature as per with the
   manufacturer's instructions and analyzed using Merck&Millipore Muse
   Cell Analyzer. Viability of ADSCs and CD146^+ cells was determined
   using Muse Annexin V & Dead Cell Kit (MCH100105). Cell suspension and
   Muse Count & Viability reagent were added to each tube, incubated for 5
   min at room temperature, and analyzed using the Merck& Millipore Muse
   Cell Analyzer.

Microarray analyses

   Total RNA was isolated from hADSCs and freshly sorted CD146^+ cells
   using Trizol reagent and purified with mirVana mRNA Isolation Kit
   (Ambion, Austin, TX, USA). The RNA integrity was determined using the
   RNA 6000 Nano Lab-on-a-Chip kit and the Bioanalyzer 2100 (Agilent
   Technologies, Santa Clara, CA, USA). Only RNA extracts with RNA
   integrity number values >6 were used for further analysis. Gene
   expression analyses were performed using the Agilent human mRNA Array.
   The array data were analyzed with the help of Capitalbiotech for data
   summarization, normalization, and quality control by using the
   GeneSpring software V13 (Agilent). To select the differentially
   expressed genes, we used threshold values of ≥2- and ≤0.5-fold change
   and a Benjamini-Hochberg-corrected P-value of 0.05. The data were
   log(2)-transformed and median-centered by genes using the Adjust Data
   function of CLUSTER 3.0 software. The data were further analyzed with
   hierarchical clustering with average linkage [110]^38. Tree
   visualization was performed by using Java Treeview (Stanford University
   School of Medicine, Stanford, CA, USA). Gene ontology (GO) pathway
   enrichment analysis was performed by WEGO
   ([111]http://wego.genomics.org.cn/cgi-bin/wego/index.pl).

Cytokine analysis

   The ADSCs and CD146^+ cells were lysed and quantified. The lysates were
   analyzed for chemokines and growth factors (80 cytokines in total)
   using a RayBio® Human Cytokine Antibody Array G5 (RayBiotech, USA),
   following the manufacturer's instructions. Data were analyzed using
   GenePix Pro 6.0.

Rat osteochondral defect model and histological examination

   Eight-week-old SD rats with a mean weight of 220 ± 20 g were used in
   this study (Figure [112]1C). Using an electric drill, osteochondral
   defects (1.5 mm diameter and 1 mm depth) were created through both the
   chondral and the subchondral bone layer of the patellar trochlear
   groove in their right legs. The joint capsule and the overlying muscle
   were closed with a suture. All rats were randomly allocated into four
   groups: defects with intra-articular injections of PBS (negative
   control), defects with intra-articular injections of ADSCs (1×10^5),
   defects with intra-articular injections of CD146^+ cells (1×10^5), and
   the sham-operated group. The knee joint samples were collected 2 weeks
   after the operation. For histological examination, the samples were
   stained with immunohistochemistry staining of HLA-ABC (Abcam, 1:100),
   IL-1β (Abcam, 1:100), IL-6 (Abcam, 1:100), IL-10 (Abcam, 1:100), H&E,
   Safranine O, and Toluidine Blue.

Multiplex cytokine assay

   Areas of the cartilage and subchondral bone with defects were selected.
   Inflammatory cytokines present in the cartilage and subchondral bone
   samples were detected following the manufacturer's instructions
   (RayBiotech, USA). Data were analyzed using GenePix Pro 6.0.

Fabrication of cell-scaffold composites

   The synthesis of the Production of the ACECM scaffold was described
   previously [113]^39. The diameter of the scaffolds was 3.5 mm and the
   height of the scaffold was 2 mm. A total number of 5×10^5 hADSCs or
   freshly sorted CD146^+ cells were seeded into ACECM scaffolds and
   cultured in DMEM/F12 for 3 days.

Scanning electron microscopy

   After being in cluture for 3 days, the microarchitecture of
   cell-scaffold composites was analyzed using the scanning electron
   microscopy (SEM) (S-2600N, Japan). Cell-scaffold composites, after
   de-hydrating in a graded ethanol series, were dried in a critical point
   dryer and coated with gold-palladium (EMS850X, USA).

Live/dead staining and confocal microscopy for cell viability/growth

   After being in culture for 3 days, the viability and growth of cells in
   the scaffold were assessed by live/dead staining following the
   manufacturer's guidelines (Sigma). The viable cells were labeled in
   green and dead cells were labeled in red. Live and dead cells were
   visualized and scored using fluorescence imaging and confocal
   microscopy (Leica Microsystems DM6000B-SP57CS).

In vivo biocompatibility and degradability

   Cell-scaffold composites were subcutaneously embedded into the back
   skin of New Zealand white rabbits (~2.0 kg) to evaluate their
   degradation and biocompatibility. At 1, 2, and 4 weeks after
   implantation, rabbits were euthanized, and photographs of the remaining
   cell-scaffold composites under the skin were taken. H&E staining was
   also carried out to evaluate the histological changes.
   Immunofluorescence staining of HLA-ABC (Abcam) was performed to
   evaluate human cell persistence overtime.

In vivo surgical procedure of full-thickness rabbit cartilage defect repair

   Sixty healthy New Zealand white rabbits (~2.0 kg) were employed for the
   in vivo cartilage defect repair experiments (Figure [114]1D). The
   region of interest was shaved and aseptically prepared for operation.
   Using an electric drill, a cartilage defect (3.5 mm diameter and 2 mm
   depth) was created through the chondral layers of the patellar
   trochlear groove in their right legs. The defects of the experimental
   group were implanted with ACECM scaffold, ADSCs & ACECM scaffold
   composites, and CD146^+ subpopulation & ACECM scaffold composites. The
   defects of the negative control group received no treatment. A
   sham-operated group was also included. Each group consisted of 12
   healthy New Zealand white rabbits. Animals were housed individually and
   given penicillin for 7 days following the operation. All rabbits were
   euthanized at the pre-determined time points (3 or 6 months). The knee
   joints were collected for further evaluation.

X-ray and magnetic resonance imaging

   The samples were first assessed using an X-ray scanner (Faxitron, USA).
   Subsequently, the magnetic resonance imaging (MRI) scans were taken
   using a 7.0 T Bruker Biospec system (Bruker Biospec, Germany).
   T2-weighted spin-echo images with fat suppression were obtained in the
   sagittal, frontal, and transverse planes. All images were scored
   independently following the Score System for MRI Evaluation [115]^40
   (Table [116]S2).

Macroscopic observations

   The cartilage defect area in the femoral condyles and the tibial
   plateau was observed and photographed. All images were scored
   independently by an experienced researcher specialized in
   musculoskeletal disease and blinded to the group assignments, following
   the International Cartilage Repair Society (ICRS) macroscopic
   evaluation guidelines (Table [117]S3).

Histology and histomorphometry

   For histological examination, tissue samples were fixed in 4% PFA,
   de-calcified in EDTA, and prepared for paraffin-embedded sections. The
   regenerated cartilage was sectioned into 6-μm slices and stained with
   H&E, Safranin-O, Toluidine blue and Sirius Red following the
   manufacturer's protocols. All images were scored independently by an
   experienced researcher specialized in musculoskeletal disease and
   blinded to the group assignments according to the ICRS histological
   assessment scale (Table [118]S4).

Statistics

   All quantitative data were analyzed using SPSS version 22.0 (SPSS,
   Chicago, IL, USA) and reported as the mean ± standard error of the
   mean. Student's t-test or one-way analysis of variance (one-way ANOVA),
   followed by the Bonferroni multiple comparison test, was performed for
   normally distributed data. A value of P < 0.05 was considered to
   indicate a statistically significant difference.

Study approval

   All animal experiments were performed strictly following the standards
   for the care and use of laboratory animals and were approved by the
   Institutional Animal Care and Use Committee of the Chinese PLA General
   Hospital.

Results

Enriched CD146^+ subpopulation retains the expression of mesenchymal stem
cell and pericyte surface markers, and has a better immune character

   CD146^+ subpopulation was sorted by MACS and the expression of stem
   cell surface markers was examined. Similar to hADSCs, CD146^+
   subpopulation expressed the cell surface markers CD73, CD90, and CD105,
   but did not express CD34, CD45, and HLA-DR (Figure [119]2A). After the
   enrichment, the percentage of the CD146^+ subpopulation increased from
   13.48% to 88.12% (Figure [120]2A). To determine the expressions of
   other pericyte surface markers in CD146^+ subpopulation,
   immunofluorescence staining and Western blotting were performed (Figure
   [121]2B-C). As shown by immunofluorescence staining, the CD146^+
   subpopulation co-expressed the other pericyte markers NG2, PDGFR-β, and
   RGS5 (Figure [122]2B). Western blotting demonstrated that the
   expression levels of these markers in CD146^+ subpopulation were higher
   than those in hADSCs before enrichment (Figure [123]2C).

Figure 2.

   [124]Figure 2
   [125]Open in a new tab

   Surface markers expression and immune characteristics of CD146^+ ADSCs
   before and after MACS enrichment. (A) Flow cytometric analysis of
   MSC-specific surface markers in CD146^+ ADSCs and donor-matched ADSCs.
   CD146+ ADSCs and donor-matched ADSCs were stained for CD34, CD45, CD73,
   CD90, CD105, CD146, and HLA-DR. (B) Immunofluorescence images for CD146
   (red), NG2 (green) RGS5 (green) and PDGFR-β (green) in CD146^+ ADSCs
   and donor-matched ADSCs. Scale bar: 20 μm. (C) Western blots analysis
   of MHC-I, MHC-II, CD40, CD80, CD86, NG2, PDGFR-β, and RGS5. MHC-II was
   not detected in either group. (D) Representative images of MHC-I and
   MHC-II immunohistochemical staining in CD146^+ ADSCs and donor-matched
   ADSCs. Scale bar: 50 μm.

   We further investigated the immune characteristics of the CD146^+
   subpopulation. The expression of MHC determines the matching of
   allografts with hosts. As validated by immunocytochemistry and Western
   blotting, the expression of MHC-I was faintly positive in CD146^+
   subpopulation, and was lower than hADSCs before sorting (Figure
   [126]2D), whereas the expression of MHC-II was negative in both ADSCs
   and the CD146^+ subpopulation (Figure [127]2D). CD40, CD80 (B7-1), and
   CD86 (B7-2) are co-stimulatory molecules, which interact with CD28 of
   T-cells and form the second signal necessary for T-cell activation. The
   absence of these molecules causes T-cell anergy. No significant
   difference was found in the expression levels of CD40, CD80, and CD86
   between the CD146^+ subpopulation and hADSCs as determined by qPT- PCR
   and Western blotting (Figure [128]2C). IDO, PGE2, IL-10, and HGF are
   molecules that have an immune suppressive effect. Although the
   expression levels of IDO in the CD146^+ subpopulation were lower than
   those in ADSCs, the expression levels of PGE2 and IL-10 were
   significantly higher in the CD146^+ subpopulation (Figure [129]S2).

CD146^+ subpopulation retains a high proliferation rate and viability after
the enrichment

   Proliferation capacity of the CD146^+ subpopulation was examined by
   morphology observation and Cell Counting Kit-8 (CCK-8) assay on day 1,
   3, and 5(Figure [130]3A-B). The results showed that both ADSCs and
   CD146^+ cells adhered to the surface and proliferated well under
   standard culture conditions (Figure [131]3A). The latent phase and
   logarithmic phase lasted from day 1-3, and the stationary phase lasted
   from day 3-5. No significant difference was observed between ADSCs and
   CD146^+ subpopulation during the entire period (Figure [132]3B). Cell
   viability (Figure [133]3E) and apoptosis (Figure [134]3C) were
   determined by flow cytometric analysis, and the percentages of cells in
   different stages were calculated (Figure [135]3D, F). The quantitative
   analysis suggested that the CD146^+ subset maintained high cell
   viability (over 95%) and a low apoptotic rate after the enrichment
   confirming the good growth properties (Figure [136]3D, F).

Figure 3.

   [137]Figure 3
   [138]Open in a new tab

   Proliferation, apoptosis, and viability of CD146^+ ADSCs before and
   after MACS enrichment. (A) Morphology of CD146^+ ADSCs and
   donor-matched ADSCs on day 1, 3, and 5. Cells were cultured at the same
   density on day 1. Scale bar: 50 μm. (B) Cell proliferation CCK8 assay
   of ADSCs and CD146^+ ADSCs. *P < 0.05, pairwise comparisons, Student's
   t-test. (C) Apoptosis in ADSCs and CD146^+ subpopulation was determined
   by flow cytometric analysis. (D) The proportion of apoptotic ADSCs and
   CD146^+ subpopulation. The proportions of live, early apoptotic, late
   apoptotic, debris, and dead cells of the CD146^+ subpopulation are
   shown in mean ± standard error, ***P < 0.05 by Student's t-test
   compared with ADSCs. (E) Viability in ADSCs and CD146^+ cells by flow
   cytometry. (F) Cell viability of ADSCs and CD146^+ subpopulation. The
   proportions of live and dead CD146^+ cells are shown in the mean ±
   standard error of the mean, ***P < 0.05 by Student's t-test compared
   with ADSCs.

CD146^+ subpopulation is distinct from hADSCs with a unique molecular
signature

   To identify the molecular signature of the CD146^+ subpopulation, we
   performed an mRNA and protein expression microarray analyses and
   compared the expression profiles of cells before and after sorting. In
   mRNA microarrays, a total of 428 differentially expressed genes were
   identified, of which 221 genes were up-regulated in CD146^+ cells, and
   208 were downregulated (Figure [139]4A). Differential gene expression
   was subjected to cluster analysis in which biological replicates in
   each group were clustered together (Figure [140]4A). Differentially
   expressed genes included immune and inflammatory genes such as IL6,
   CXCL2, IL1RL1, IL17RD, IL18, and IL26 (Figure [141]4B). Gene ontology
   (GO) classification of genes by biological processes showed
   differentially expressed genes were involved in multicellular
   organismal processes, such as chemotaxis, wound healing, blood vessels,
   and inflammatory processes (Figure [142]4C). Protein expression of
   ADSCs and CD146^+ subpopulation was tested by using antibody
   microarrays. Among 80 chemokines and growth factors, 11 differentially
   expressed proteins were identified (Figure [143]4D). Differentially
   expressed chemokines included immune and inflammatory proteins such as
   IL6, IL12 and IL16. GO classification showed that differentially
   expressed proteins play a role in chemotaxis and cell division (Figure
   [144]4E-F). The combined gene and protein expression results indicated
   that, compared to hADSCs, the CD146^+ subpopulation was a distinct
   population and had a molecular signature in inflammation functions
   before sorting.

Figure 4.

   [145]Figure 4
   [146]Open in a new tab

   Gene and protein expression of CD146^+ ADSCs before and after MACS
   enrichment. (A) Heatmap showing transcription expression profiles of
   ADSCs and the CD146^+ subpopulation. Differentially expressed genes are
   shown. Each block represents the relative transcript level of an
   individual gene. Each column represents the profile of an individual
   group. (B) List of inflammation related genes that were significantly
   changed in CD146^+ cells. (C) GO classification of genes by biological
   process. (D) Cytokine expression of ADSCs and CD146^+ cells. Samples
   were collected from cell protein. Heatmap showing cytokine expression
   profiles of ADSCs and CD146^+ cells. (E) GO classification of proteins
   by molecular function. (F) KEGG pathway of proteins.

CD146+ subpopulation reduces inflammation of the articular cartilage during
the early stages of cells injection

   To elucidate the cellular functions in vivo, we employed a rat
   osteochondral defect model obtained by intra-articular injections of
   cells, and formulated four groups: defects with intra-articular
   injections of PBS (negative control), ADSCs, and CD146^+ subpopulation,
   and the sham-operated group (Figure [147]5A). Macroscopic observation
   showed that the defect area could not be totally repaired in two weeks
   after injection in all the groups (Figure [148]5A). Immunohistochemical
   staining of HLA-ABC specially marked the location of the human cells in
   the early stages of cell implantation (Figure [149]5B). Results showed
   that both hADSCs and CD146^+ subpopulation were recruited into the
   defect area. Histologically, although a time span of 2 weeks was too
   short for the repair, intra-articular injections of CD146^+
   subpopulation led to better improvements of the quality of
   osteochondral repair, with less inflammatory cells in the boundary
   between healthy tissue and the defected area (Figure [150]5C).

Figure 5.

   [151]Figure 5
   [152]Open in a new tab

   Role of CD146^+ ADSCs in cartilage microenvironment after joint cavity
   injection. (A) Experimental design for cell transplantation and
   macroscopic analyses of femoral condyle cartilage. Rats were randomly
   allocated to four groups: defects treated with PBS (negative control),
   defects treated with ADSCs, defects treated with CD146^+ cells, and the
   sham-operated group. At 2 weeks, rats were euthanized, and the distal
   femora were harvested. (B) Immunohistochemical staining of HLA-ABC.
   Scale bar: 100 μm. (C) Histological analysis of the defected area by
   H&E, Safranin O, and Sirius red staining. Black solid arrows denote the
   repair interface. Red solid arrows denote the depth of the repaired
   cartilage. HC, host cartilage; D, defect area; RC, repaired cartilage.
   Scale bar: 100 μm. (D) Analysis of inflammation related cytokine
   expressions. Results represent the mean ± standard error of the mean;
   the sample number was three. *P < 0.05, **P < 0.01. (E)
   Immunohistochemical staining of IL-1β, IL-6, IL-10 and TNF-α. Black
   solid arrows denote the positive expression of IL-6 in the repair
   interface Scale bar: 100 μm.

   Inflammatory cytokines in articular cartilage and subchondral bone were
   further investigated. The expression level of IL-6 was significantly
   higher in the hADSCs group, than in the CD146^+ group which was more
   similar to that in normal cartilage (Figure [153]5D). Also, the
   expression of cytokines such as IL-6, IL-1β, TNF-α, and IL-10 in the
   defect area was evaluated histologically. Two weeks after the
   injection, there was no significant difference in the expression of
   IL-1β, TNF-α and IL-10, while the expression of IL-6 was still present
   on the surface of the defect area in the negative control and ADSCs
   injected groups. Less expression of IL-6 was observed in the defect
   area of CD146^+ cells injected groups (Figure [154]5E). Thus, the
   CD146^+ subpopulation could reduce inflammation during the early stages
   of cells injection to promote cartilage repair.

CD146^+ cell-scaffold composites resulted in less subcutaneous inflammation

   Given that CD146^+ subpopulation was associated with inflammation
   moderation of the cartilage microenvironment during the early stages of
   implantation, we developed ACECM scaffold composites for long-term
   cartilage repair. Same numbers of ADSCs and CD146^+ subpopulation were
   seeded into ACECM scaffolds and cultured for 3 days in vitro (Figure
   [155]6A). Macroscopic photographs of the three groups are displayed
   Figure [156]6B. Scanning electron microscopy (SEM) images show the
   porous structure of the ACECM (Figure [157]6C). The inner walls of the
   evenly distributed pores increase the surface area and are suitable for
   cell adhesion. Both ADSCs and CD146^+ subpopulation were located in the
   inner wall of the pores, maintained their phenotype, and performed
   normal function after 3 days of culturing (Figure [158]6C).
   Furthermore, cell viability was measured by live/dead cell staining
   (Figure [159]6D). Both CD146^+ subpopulation and ADSCs proliferated and
   grew well in the three-dimensional porous scaffold and most viable
   cells exhibited a uniform distribution (Figure [160]6D). These results
   suggested that the ACECM is not toxic to cells and is suitable for cell
   growth as a useful biocarrier material for cartilage repair.

Figure 6.

   [161]Figure 6
   [162]Open in a new tab

   Fabrication and biocompatibility of cell-scaffold composites. (A)
   Experimental design of the cell-scaffold composites. ADSCs and CD146^+
   cells were seeded into an ACECM scaffold for 3 days. (B) Macroscopic
   features of the ACECM scaffold and two cell-scaffold composites. (C)
   SEM images of the ACECM scaffold and two cell-scaffold composites. (D)
   Live/dead cell analysis for the two cell-scaffold composites.
   Representative images show dead cells (red), live cells (green), and
   reconstruction images of the two cell-scaffold composites. (E)
   Photographs of the ACECM scaffold and two cell-scaffold composites
   subcutaneously implanted in the back of rabbits after 1 week, 2 weeks,
   and 4 weeks (the circle indicates the tissue surrounding the scaffold).
   (F) Immunofluorescent staining of HLA-ABC of the tissues surrounding
   the implant sites after 1 week, 2 weeks, and 4 weeks. Scale bar: 50 μm.
   (G) H&E staining of the tissues surrounding the implant sites after 1
   week, 2 weeks, and 4 weeks. Scale bar: 100 μm.

   To further explore the in vivo degradation and biocompatibility of the
   composites, three types of cell-scaffold composites (ACECM scaffold,
   ADSCs & ACECM scaffold composites, and CD146^+ subpopulation & ACECM
   scaffold composites) were subcutaneously implanted into the back skin
   of rabbits, and their role in modulating subcutaneous inflammation was
   investigated (Figure [163]6E-G). Macroscopic photographs showed that in
   1, 2, and 4 weeks after implantation, cell-scaffold composites adhered
   to a layer of connective tissue and were degraded gradually in vivo
   (Figure [164]6E). Survival of human cells overtime was assessed by
   immunofluorescent staining of HLA-ABC. The results showed that ADSCs
   and CD146^+ subpopulation retained high survival in 1, 2, and 4 weeks
   after implantation (Figure [165]6F). As per H&E staining, the
   appearance of inflammatory cells in the first week suggested an acute
   inflammatory response in all three groups (Figure [166]6G). From 2 to 4
   weeks, the number of inflammatory cells in all three groups decreased
   gradually, reflecting the reduction in inflammatory response (Figure
   [167]6G). During the entire period of observation, the lowest number of
   inflammatory cells was observed in the CD146^+ subpopulation & ACECM
   scaffold composite group, confirming that this scaffold composite
   caused the least subcutaneous inflammation.

CD146^+ subpopulation in combination with the ACECM scaffold improves
cartilage regeneration

   To verify the role of cell-scaffold composites in long term cartilage
   repair, we implanted them into the trochlear groove cartilage defect
   areas in rabbits. X-ray images revealed that there was no osteophyte
   formation and heterotopic ossification in any of the five groups during
   the experimental period (Figure [168]7B, Figure [169]S3). At 3 months,
   new cartilage tissue was observed in the ACECM scaffold, ADSCs & ACECM
   scaffold composite, and CD146^+ subpopulation & ACECM scaffold
   composite implanted groups by high-resolution MRI of the fresh whole
   knee joints, but immature cartilage signal was also present (Figure
   [170]7C). At 6 months, MRI images of the ADSCs & ACECM scaffold
   composite and CD146^+ subpopulation & ACECM scaffold composite
   implanted groups were almost entirely filled with new cartilage tissue
   better than the ACECM scaffold implanted group (Figure [171]7C). During
   this period, no new tissue was found in the negative control group
   (Figure [172]7C). MRI score analysis also confirmed these results. As
   shown in Figure [173]7D, the score in CD146^+ subpopulation & ACECM
   scaffold composite implanted groups was higher than the other groups at
   3months. At 6 months, both ADSCs & ACECM scaffold composite and CD146^+
   subpopulation & ACECM scaffold composites implanted groups achieved a
   high score that was comparable to the native groups (Figure [174]7D).

Figure 7.

   [175]Figure 7
   [176]Open in a new tab

   Radiography assessment of the cartilage in rabbits with cell-scaffold
   composites treatment. (A) Experimental design for the cartilage repair.
   The rabbits were randomly allocated into five groups: defects without
   treatment (negative control), defects treated with ACECM scaffold,
   defects treated with ACECMs and ADSCs, defects treated with ACECMs and
   CD146^+ cells, and native group. (B) X-ray images of rabbit knees 3 and
   6 months after surgery. (C) MRI of rabbit knees 3 months and 6 months
   after surgery. Red arrow indicates the defected area. The inset in the
   bottom left corner of each T2 mapping image shows the cartilage labeled
   by pseudo-color. (D) MRI scoring system for evaluation of cartilage
   repair after 3 and 6 months. Data are expressed as mean ± SD, *P <
   0.05, **P < 0.01, ***P < 0.0001.

   The entire knee joint was dissected for macroscopic evaluation. No
   apparent inflammation and synovial hyperplasia were observed in any of
   the groups during the 6 months (Figure [177]8A). New cartilage
   generally formed from the edge of the defects in the ACECM scaffold and
   two cell-scaffold composite implanted groups from 3 to 6 months (Figure
   [178]8A). Generally, the shape of the new cartilage was better at 6
   months compared with 3 months (Figure [179]8A). The regenerated
   cartilage in the CD146^+ subpopulation & ACECM scaffold composite
   implanted group was almost similar to native cartilage after 6 months
   (Figure [180]8A). In the negative control group, the defect did not
   regrow during this period (Figure [181]8A). Consistent with the
   macroscopic results, the International Cartilage Repair Society (ICRS)
   macroscopic evaluation score for regenerated cartilage in the CD146^+
   subpopulation & ACECM scaffold composite implanted group was better
   than that of the other groups at 3 and 6 months (Figure [182]8B-C,
   Figure [183]S4).

Figure 8.

   [184]Figure 8
   [185]Open in a new tab

   Macroscopic and histological evaluation of cartilage repair after
   cell-scaffold composite treatment. (A) Macroscopic observations of
   rabbit knees (distal femur and tibial plateau) 3 and 6 months after
   surgery. (B) (C) ICRS scoring system for macroscopic evaluation of
   cartilage repair after 3 months (B) and 6 months (C). Data are
   expressed as mean ± SD, *P < 0.05, **P < 0.01, ***P < 0.0001. (D)
   Histological analysis of the cartilage defect after 3 and 6 months by
   H&E and Sirius Red staining. Black solid arrows denote the repair
   interface. Red solid arrows denote the depth of the repaired cartilage.
   HC, host cartilage; RC, repaired cartilage. Scale bar: 200 μm. (E) (F)
   ICRS scoring system for histological evaluation of cartilage repair
   after 3 months (E) and 6 months (F).

   The thickness and structure of new cartilage in the defected area were
   visualized histologically (Figure [186]8D-F, Figure [187]S5-8). The
   chondrocyte-like cells were visualized in the defected area in the
   ACECM, ADSCs & ACECM, and CD146^+ subpopulation & ACECM scaffold
   composite implanted groups. Safranine O and Toluidine Blue stainings
   were negative in the defected area and positive in the regenerated
   hyaline cartilage. Generally, as the repair period progressed, the
   defect was gradually filled with new cartilage (Figure [188]8D-F,
   Figure [189]S5-8). The new cartilage layer was thin in the ACECM
   scaffold implanted group compared with the ADSCs & ACECM, and CD146^+
   subpopulation & ACECM scaffold composite implanted groups. However, no
   new cartilage layer was found in the negative control group at both 3
   and 6 months (Figure [190]8D-F, Figure [191]S5-8). At 6 months, the
   thickness of the new cartilage layer in the CD146^+ subpopulation &
   ACECM scaffold composite implanted group was similar to that of the
   native cartilage, with less void space and a regular surface. Sirius
   Red staining also highlighted a tangled arrangement of collagen fibers
   in the CD146^+ subpopulation & ACECM scaffold composite implanted group
   at 6 months (Figure. 8D-F, Figure [192]S6-9). Better regeneration of
   the cartilage in the CD146^+ subpopulation & ACECM scaffold composites
   implanted group was also confirmed by the International Cartilage
   Repair Society (ICRS) macroscopic evaluation score.

Discussion

   It has long been known that MSCs comprise a heterogeneous population,
   which impedes their application in regenerative medicine [193]^22.
   Studies found that some subpopulations have enhanced capacity for
   cartilage formation. For example, Dickinson et al. reported the
   subpopulation with the receptor tyrosine kinase-like orphan receptor 2
   (ROR2) as a cell surface marker is effective at making cartilage. The
   application of subpopulations is emerging as a novel idea for cartilage
   repair [194]^41.

   As a surface marker expressed by pericytes and the smooth muscle cells
   of blood vessels, CD146 plays a critical role in cell adhesion,
   embryonic development, immune response, angiogenesis, and cancer
   [195]^42^-[196]^45. Previous studies have shown that CD146 defines a
   functional subset of progenitor cell populations and exhibit higher
   developmental potentials [197]^27^, [198]^46^-[199]^47. In vivo,
   CD146^+ cells surround endothelial cells populating the vascular
   intima. With specific adhesion and migration properties, CD146^+ cells
   can regulate blood vessel stability/integrity as well as the
   proliferation and motility of adjacent endothelial cells [200]^48. Both
   in vitro and in vivo studies provided evidence that CD146+ perivascular
   cells, as constituents of MSCs, and play a critical role during wound
   healing [201]^49. For example, increased number of CD146^+ cells by
   activation of PDGF-BB/ PDGFRβ signaling pathway has been reported
   during wound healing in vivo [202]^50. CD146^+ cells have also been
   shown to promote wound healing by secreting high levels of cytokines
   such as KGF, vascular endothelial growth factor (VEGF), heparin
   binding-epidermal growth factor (HB-EGF) and basic-fibroblast growth
   factor (bFGF) [203]^51. CD146+ cells isolated from human lipoaspirate
   showed immunomodulatory effect during bone formation [204]^52. In
   another study, CD146^+ cells have been shown to induce increased cord
   formation [205]^53. In a degenerated intervertebral disc model, CD146^+
   MSCs showed greater migration potential for re-population [206]^54.
   However, the role of CD146^+ cells as a seed cell type in cartilage
   tissue engineering is unclear.

   In this study, a CD146^+ subpopulation of hADSCs was selected, and
   applied for cartilage repair as evidence from both in vitro and in vivo
   experiments supports its role in cartilage repair. The expressions of
   MSC related surface markers were almost unchanged after the enrichment.
   Pericytes as progenitor cells ubiquitously express surface markers such
   as NG2, PDGFR-β, and RGS5 in almost all organs [207]^27^, [208]^55.
   Consistent with previous reports, the CD146^+ population from hADSCs
   expressed high levels of other pericyte markers, was distinct from
   hADSCs, and maintained their progenitor cell character. Immunogenicity
   affects the outcome of cell transplantation. MSCs can avoid allogeneic
   rejection, prolong graft survival, and induce tolerance in humans and
   in animal models. When CD146^+ cells were sorted from human adipose
   tissue for bone formation in mice, no immunological rejection was
   observed [209]^52. MHC class II proteins are potent alloantigens. Under
   non-inflammatory conditions, human MSCs are MHC-II negative, supporting
   a role for MSC in having reduced immunogenicity through the control of
   alloantigen expression [210]^56^-[211]^60. The expression of MHC-I in
   the CD146^+ subpopulation is significantly lower than in hADSCs with no
   MHC-II expression, supporting a role for the CD146^+ subpopulation with
   reduced immunogenicity through the control of alloantigen expression.
   Collectively, the CD146+ population has immune characteristics to avoid
   allogeneic rejection.

   Previous studies found that CD146 was associated with proliferation and
   expressed at high levels in nearly all clonal MSCs, while only a
   minority of nonclonal MSCs expressed this marker [212]^31. In this
   study, we observed high proliferation rate, cell viability, and low
   apoptosis verifying the cell survival capability of CD146^+ cells, and
   supporting their suitability for transplantation. It was important to
   elucidate the characteristic features of CD146^+ cells at the molecular
   level. Our microarray data highlighted a variety of functions of
   CD146^+ cells in chemotaxis, wound healing, blood vessels, and
   inflammatory processes, confirming that the CD146^+ population is
   functionally distinct from the hADSCs.

   Cartilage defects typically lead to posttraumatic inflammation and
   represent a significant challenge in cartilage repair [213]^61. It has
   been shown that MSCs can promote tissue repair by modulating
   inflammation [214]^62. In vitro, both gene and protein results revealed
   a differential expression profile of inflammation related cytokines
   between CD146^+ cells and hADSCs. To investigate the specific cellular
   functions in the cartilage microenvironment in vivo, we injected
   CD146^+ cells into the joint cavity. In the defect area, CD146^+ cells
   did not lead to higher expression levels of IL-6 compared with hADSCs.
   This result is consistent with a previous report showing that a CD146^+
   subpopulation from human umbilical cord cells provided an
   anti-inflammatory protective microenvironment by suppressing IL-6
   [215]^34. Another study also reported that CD146^+ cell implantation
   reduced Ly-6G^+ and F4/80^+ cell infiltration in bone [216]^52. Thus,
   CD146^+ population after enrichment plays a better role in modulating
   inflammation.

   The clinical applications of MSCs is often limited by their poor
   viability at the site of injury caused by the unfavorable
   microenvironment and to anoikis driven by the loss of cell adhesion
   [217]^63. To increase the viability of cells, we fabricated a cell
   ACECM scaffold composites to create a proper microenvironment for cell
   adhesion. For long-term cartilage regeneration, ACECM is the best
   biomaterial to mimic native cartilage and drive tissue homeostasis and
   regeneration because of the presence of bioactive molecules [218]^64.
   Also, ACECM is biodegradable and does not elicit adverse immune
   responses [219]^64. In this study, several findings supported good
   biocompatibility of cell ACECM scaffold composites. First, SEM images
   showed the loose and porous structure of ACECM that is suitable for
   cell adhesion. ADSCs and CD146^+ cells were located on the inner wall
   of pores, maintained their phenotype, and functioned normally after
   culturing. Second, CD146^+ cells and ADSCs proliferated and grew well
   in the three-dimensional porous scaffold, and the most viable cells
   exhibited a uniform distribution. Third, when the degradability of the
   cell-scaffold composites in vivo was tested by subcutaneous
   implantation in rabbits, they displayed good biocompatibility without
   any visible foreign body reaction. Finally, CD146^+ subpopulation
   survived even 4 weeks after implantation confirming that ACECM scaffold
   created a proper microenvironment for cell adhesion. The lower number
   of inflammatory cells confirmed that the CD146^+ cells caused a more
   significant reduction in subcutaneous inflammation than the more
   heterogeneous hADSCs. These results were consistent with the previous
   finding that CD146^+ cells combined with collagen scaffold did not
   induce an early inflammatory infiltrate during bone formation [220]^52.

   In the rabbit implantation experiment, the outcome observed in the
   CD146^+ subpopulation & ACECM scaffold composite implanted group was
   superior to all other groups as confirmed by imaging, macroscopic
   observation, and histological examination. The regenerative capacity of
   tissues depends on the degree of the inflammatory response [221]^65. We
   hypothesized that CD146^+ subpopulation would play a superior role in
   inhibiting inflammation and promoting long term outcome. ACECM can
   stimulate growth and proliferation, and ensure the normal functioning
   of CD146^+ subpopulation. Enhanced proliferation of the CD146^+
   subpopulation can offset the dysfunction caused by the insufficient
   number of cells. Also, direct differentiation of MSCs into chondrocytes
   is one of the key factors for cartilage repair. In this context, the
   differentiation ability of the CD146^+ subpopulation has been
   demonstrated in previous studies [222]^27^, [223]^66^-[224]^68. CD146^+
   cells can directly differentiate into chondrocytes and promote
   cartilage repair. However, CD146 is not a specific chondrogenesis
   marker, although some studies have shown that CD146^+ subset has better
   chondrogenic differentiation ability [225]^67. It is of note that
   CD146^- subset can also differentiate into chondrocytes [226]^54.
   CD146^- cells are also heterogeneity and contain progenitors cells of
   MSC. For example, CD34+ cells, resides in the outmost layer of blood
   vessels, also natively express MSC markers and give rise in culture to
   progenitors cells of MSC [227]^69. The regulation of chondrogenic
   differentiation in CD146^+ and CD146^- cells remains to be further
   investigated.

   The quantity and stability of cells are important factors that affect
   subpopulation-based therapy. CD146+ cells sorted from fetal and adult
   muscle, midterm placenta, fetal skin, fetal pancreas, bone marrow, and
   adipose tissue can retain the phenotype, exhibit osteogenic,
   chondrogenic, and adipogenic potentials and express MSC and pericyte
   markers [228]^27. For example, muscle-derived CD146+ cells stably
   expressed pericyte markers such as NG2, CD146, and α-SMA after 4, 8, or
   14 passages [229]^27. CD146+ cells from mesenchymoangioblast can be
   cultured in the presence of platelet-derived growth factor (PDGF)-BB
   for up to 12 passages with gradual senescence observed during 8-12
   passages [230]^70. In our study, we used cells for in vivo cartilage
   repair without long term culture in vitro. Compared with bone marrow
   MSCs, large numbers of ADSCs can be obtained from lipoaspirate in one
   attempt with less pain. In the rat model, CD146^+ cells were injected
   into the joint cavity immediately after MACS enrichment without in
   vitro culture. In the rabbit model, CD146^+ cells were cultured 3 days
   before in vivo implantation. In future clinical applications,
   sufficient numbers of CD146^+ subpopulation can be obtained without
   requiring serial sub-culturing in vitro. Thus, subpopulation-ased
   strategy in this study is more feasible for clinical translation.
   Another important issue that needs to be explored in the future is the
   variability of CD146^+ cells between donors to enable selection of the
   proper subpopulation for therapy.

Conclusions

   Our study highlights the importance of a subpopulation-based MSCs
   therapy in regenerative medicine. Although it has long been known that
   MSCs are heterogeneous and subpopulations vary functionally, the
   precise roles of different subpopulations in cartilage tissue repair
   has remained elusive. Our ultimate goal is to optimize cartilage
   structure and function utilizing specific properties of MSC subtypes
   and make tissue engineering more effective. Thus, uncovering the
   functional characteristics of specific subtypes of MSCs is a new
   direction for cell-based tissue engineering research and can
   potentially further the development of precision medicine.

Supplementary Material

   Supplementary figures and tables.
   [231]Click here for additional data file.^ (1.2MB, pdf)

Acknowledgments