Divergent effects of distinct perivascular cell subsets for intra-articular cell therapy in posttraumatic osteoarthritis

2.1 Animals

All animal studies were performed with institutional Animal Care and Use Committee approval within Johns Hopkins University (JHU), complying with all relevant ethical regulations. Pdgfrα-CreER^T2 mice were a kind gift from the Bergles laboratory at JHU and are commercially available (stock No.: 018280, Jackson lab). mT/mG mice were purchased from Jackson lab (stock No.: 007576). Pdgfrβ-CreER^T2 mice were purchased from Jackson lab (stock No.: 029684). Pdgfrα-CreER^T2 or Pdgfrβ-CreER^T2 mice were crossed with mT/mG mice to generate Pdgfrα-CreER^T2; mT/mGor Pdgfrβ-CreER^T2; mT/mG mice which were used for all experiments. Tamoxifen (TM; T5648; Sigma) was provided by intraperitoneal injection as per previously validated protocols to mixed gender, 7 weeks old Pdgfrα-CreER^T2; mT/mG mice or Pdgfrβ-CreER^T2; mT/mG mice 2 weeks before cell isolation.^{13, 23} TM was dissolved in sunflower seed oil. Tail genomic DNA was used for genotyping.

2.2 Cell isolation

Pdgfra and orPdgfrβ-CreER^T2-mT/mG mice were administered TM 2 weeks in advance of cell isolation. Inguinal fat pads from 10 mice per genotype were dissected, pooled, minced, and digested with collagenase Type 2 (Worthington), 0.5% bovine serum albumin and α-minimum essential medium (Gibco) for 1.5 h at 37°C. mGFP⁺ cells were isolated with LSR II (BD Biosciences) using FACS Diva software (BD Biosciences) and Flow Jo v10 software (Tree Star, Inc.). Cell debris was excluded based on size characterizationand mGFP⁺ cells were collected. Cells were cultured for 2–3 passages prior in vivo administration. Cells were cultured in Dulbecco's modified Eagle's medium (DMEM) at 37°C in a humidified atmosphere containing 95% air and 5% CO₂ in DMEM (Thermo Fisher Scientific, Inc.), with 1% fetal bovine serum (Thermo Fisher Scientific), and 1% penicillin/streptomycin. Medium was changed every 3 days unless otherwise stated. Cells were characterized by flow cytometry after 2 weeks culture expansion. A total of 1 × 10⁶ trypsinized cells were incubated with anti-αSMA-Alexa Fluor 647, anti-Nestin-Cy7, anti-stem cell antigen 1 (Sca-1)-Alexa Fluor 647, CD146-Alexa Fluor 647, and CD34-BV421.

2.3 Destabilization of the medial meniscus

Destabilization of the medial meniscus (DMM) or sham surgery was performed at the age of 10 weeks in mixed gender C57BL/6J mice, in similarity to prior reports.²⁴ Equal numbers of male and female animals were distributed across treatment groups. Briefly, left hindlimbs were disinfected with povidone-iodine and 70% ethanol, and buprenorphine SR (1 mg/kg) was injected subcutaneously. A 1 cm longitudinal incision was made on the medial aspect of the knee joint under general anesthesia using 2% isoflurane. Blunt dissection of the joint capsule along the medial side of the patellar ligament was performed to expose the medial menisco-tibial ligament (MMTL). The infrapatellar fat pad (IFP) was exposed, displaced laterally, and the MMTL was transected using a 11-blade scalpel to destabilize the medial meniscus. The medial joint capsule and the skin were next closed with 5-0 prolene suture (Ethicon). Sham surgery was performed with a similar surgical approach with visualization, but without transection of the MMTL. One week after surgery, either 1 × 10⁶ Pdgfra⁺ or Pdgfrβ⁺ cells were injected into the intra-articular space. Equal volume of phosphate-buffered saline (PBS) was injected as a control. In total, 40 mice, 5 males, and 5 females pertreatment group, underwent surgery. Groups included (1) sham operated, (2) DMM operated, PBS treated (control), (3) DMM operated, Pdgfra⁺ cell treated, and (4) DMM operated, Pdgfrβ⁺ cell treated. Eight weeks after DMM or sham surgery, animals were sacrificed andhindlimbs were harvested for analysis.

2.4 Histology and histomorphometry

Stifle joints were fixed in 4% paraformaldehyde for 24 h, decalcified with 14% ethylenediaminetetraacetic acid for 14 days, and embedded in OCT compound (Sakura). Sagittal sections of the stifle joint were prepared at 6 µm thickness with a Cryofilm type 3c (SECTION-LAB). Inguinal fat pads from donor Pdgfra orPdgfrβ-CreER^T2-mT/mG mice were also dissected, embedded in OCT, and cryosectioned at 30 μm thickness. Routine safranin O/fast greenstaining of stifle joints section was performed with methods adopted from past work.^25-27

For immunofluorescent immunohistochemistry, sections were washed in 1× PBS, blocked in 5% normal goat serum (S-1000, Vector Labs) for 30 min, and incubated with primary antibodies specific for ACAN (1:100; ab3778; Abcam), COL10 (1:100; ab58632; Abcam), CD31 (1:100; ab28364; Abcam)and scleraxis (1:100; ab58655; Abcam) at 4°C overnight. Sections were then incubated with Alexa Fluor 647-conjugated secondary antibodies (1:200; ab150083; Abcam) or DyLight 594 (1:100; DI-1594; Vector Labs), and mounted with mounting medium containing DAPI (H-1500; Vector Labs; Table S1). Immunofluorescence images were acquired on a Leica DM 6B or LSM 880 FCS confocal microscope (Carl Zeiss).

2.5 Microcomputed tomography imaging

Microcomputed tomography (μCT) analysis of the tibial and femoral subchondral bone was performed using a SkyScan1175 system (Bruker) with the following settings: voltage, 65 kVp; current, 153 μA; resolution, 1.0-mm aluminum filter 10 μm/pixel. The images were reconstructed and analyzed using NRecon v1.7 and CTAn v1.20, respectively. XYZ axes were adjusted by Dataviewer V1.5. 3-Dimensional histomorphometric analysis was performed on cross-sectional images of the tibial and the femoral subchondral bone. We defined the region of interest as the whole subchondral bone medial and lateral compartments and used 15 consecutive images from the medial and lateral tibial plateau for 3-dimensional reconstruction and analysis. Two 3-dimensional structural parameters were analyzed; bone volume density (BV/TV) and bone mineral density (BMD).

2.6 OARSI scoring of the knee joints

Six frozen semi-serial sagittal sections with 36 µm intervals were prepared from the medial compartment of each knee joint to represent the weight-bearing area of the distal femur and proximal tibia.²⁸ Safranin O/fast green stained sections were analyzed for cartilage injury using the OARSI scoring system.²⁹ A range of 0–24 was determined according to the formula: score = grade (G1 to G6) × stage (S1 to S4). The scoring was done by three different authors (TS, SN, and MC). The average scores of both femur and tibia were summed.

2.7 Statistical analysis

Statistical analyses were performed in IBM SPSS Statistics 16.0. Quantitative data are expressed at mean ± SD. A two tailed the Student t test was used for two-group comparisons, and a one-way Analysis of variance test was used for comparisons of three or more groups, followed by Tukey's post hoc test. *p < .05 and **p < .01 were considered significant.

3.1 Isolation of Pdgfrα⁺ and Pdgfrβ⁺ cells from mouse subcutaneous adipose tissue

To precisely define the identity of perivascular cell subsets, we used inducible mouse reporter systems for Pdgfrα and Pdgfrβ, and validated cell identity. Each Cre line was crossed with mT/mG reporter animals, which express membranous TdTomato in all cells untilCre recombination occurs when the TdTomato cassette is excised, and membranous mGFP is expressed.^30-32 Perivascular cell identity was assessed in the resulting Pdgfrα^mT/mG and Pdgfrβ^mT/mG reporter animals after exposure to TM.^{13, 33} Consistent with prior reports, Pdgfrα^mT/mG reporter activity highlighted perivascular cells within the tunica adventitia, along with a portion of cells within a pericytic location,¹³ while Pdgfrβ^mT/mG reporter activity highlighted all pericytes and vascular smooth muscle cells.²² After tissue dissociation, by flow cytometry Pdgfrα⁺ cells comprised 14.5% of total viable cells, while Pdgfrβ⁺ cells comprised on average 11% of total viable cells (Figure 1). Pericyte and stem cell markers were assessed among Pdgfrα⁺  and Pdgfrβ⁺ cell fractions (Figure S1). The expression of Sca-1 expression was high across both cell preparations (90.1% and 89.4%, respectively). In contrast, αSMA and CD146 expression was enriched among the Pdgfrβ⁺ cell fraction (89.91% and 71.88% among Pdgfrβ⁺ cells and 12.75% and 4.06% among Pdgfrα⁺ cells).

To address the therapeutic potential of either Pdgfrα⁺ or Pdgfrβ⁺ expressing perivascular cells, we performed intra-articular injection of either Pdgfrα⁺ or Pdgfrβ⁺ perivascular cell subsets (Figures 1-5). Briefly, we injected either FACS purified Pdgfrα⁺ or Pdgfrβ⁺ adipose tissue perivascular cells into the stifle joint after DMM (Figure 1). Cells were prepared from inguinal fat pads of either Pdgfrα^mT/mG or Pdgfrβ^mT/mG reporter animals. Cells were injected as a one-time dose in equal numbers (1M total cells) and compared to PBS control injection. All knee joints were analyzed at 8 weeks after DMM.

3.2 Pdgfrα⁺ and Pdgfrβ⁺ cell engraftment in the stifle joint

Differences in Pdgfrα⁺ or Pdgfrβ⁺ cell engraftment and persistence were next evaluated in our model (Figure 2). Pdgfrα⁺ cells were found superficially within the articular cartilage as well as menisci, while Pdgfrβ⁺ cells were more widely distributed within all layers of cartilage as well as subchondral bone (Figure 2, upper rows). As expected, no GFP⁺ cells were identified in control tissues (Figure 2, lower rows).

We further examined the distribution of Pdgfrα⁺ or Pdgfrβ⁺ cells within the injected joint, examining in detail the meniscus, medial cruciate ligament (MCL), IFP and joint capsule (Figure 3). After DMM surgery, mGFP⁺ cells were found in the meniscus with relatively similar frequency and distribution across both Pdgfrα⁺ and Pdgfrβ⁺ cell treated joints. Interestingly, ACAN immunostaining of the meniscus showed significant changes after cell therapy. Consistent with prior reports,^{34, 35} DMM operated animals showed a reduction in meniscal ACAN immunostaining in comparison to sham operatd controls (Figure 3A, left). Conversely, both Pdgfrα⁺ and Pdgfrβ⁺ cell treated joints showed a relative increase in ACAN immunoreactivity in areas around cell incorporation within the meniscus (Figure 3A, right).

Incorporation of injected cells into the MCL was also observed (Figure 3B). Here, DMM ligamentocytes changed from a consistent spindled shape with elongated nucleus to a more rounded cell shape (Figure 3B, left). This change in morphology was associated with a reduction in scleraxis (SCX) immunoreactivity, consistent with prior reports.³⁶ Significant incorporation of injected Pdgfrβ⁺ but not Pdgfrα⁺ cells was found within and around the MCL (Figure 3B, right). As well, SCX expression was partially restored among ligamentocytes upon either Pdgfrα⁺ or Pdgfrβ⁺ cell treatment.

Incorporation of injected cells was also found within perivascular areas of the IFP (Figure 3C). This was most conspicuous among larger microvessels of the IFP, and seen among both Pdgfrα⁺ and Pdgfrβ⁺ cell injected joints. Finally, incorporation of both Pdgfrα⁺ and Pdgfrβ⁺ fluorescent cells was found within the synovial membrane of treated joints (Figure 3D).

3.3 Subchondral bone sclerosis after Pdgfrα⁺ or Pdgfrβ⁺ cell therapy

Changes in subchondral bone after cell therapy were next examined by μCT (Figure 4). MicroCT reconstructions of femoral and tibial subchondral bone demonstrated a clear, qualitative increase in bone after DMM, which was most apparent in the medial compartment (Figures 4A,B and 4D). Cell therapies, especially Pdgfrβ⁺ cell therapy, showed a diminution of this effect. These changes were quantified separately in both the medial (Fiure 4B–E) and lateral aspects of the joint (Figure 4G–J), and separately for both femoral and tibial subchondral bone. As expected, DMM induced a significant increase in BMD within femoral and tibial subchondral bone on both medial and lateral aspects. DMM surgery led to increase in BV/TVs, which was limited to the medial tibial subchondral bone (Figure 4E). Remarkably, Pdgfrβ⁺ cell therapy completely prevented subchondral bone changes associated with DMM, including a complete reversal of any increase in BMD or BV/TV across all regions of interest. In contrast, no significant change in subchondral bone was found with Pdgfrα⁺ cell therapy in comparisons to PBS control injection. All analyses were stratified by gender, with similar results (Figure S2). Thus, Pdgfrβ⁺ but not Pdgfrα⁺ cell therapy led to improved quantitative metrics of subchondral bone sclerosis after DMM.

3.4 Histologic appearance of articular cartilage after Pdgfrα⁺ or Pdgfrβ⁺ cell therapy

Sagittal histologic sections of the knee joint were stained with safranin O/fast Green (Figure 5). Typical degenerative changes to the femoral and tibial articular cartilage were observed among PBS control-treated animals after DMM in comparison to sham surgery (Figure 5A, left hand images). Importantly, a cell dependent effect on arthritis progression was found among either Pdgfrα⁺ or Pdgfrβ⁺ perivascular cell therapy (Figure 5A, right hand images). Pdgfrβ⁺ cell injection led to a significant improvement in the appearance of the femoral and tibial articular cartilage, with less prominent loss of safranin O staining among superficial articular cartilage. In contrast, Pdgfrα⁺ cell therapy led to a less dramatic improvement in histologic appearance at 8 weeks after intra-articular injection. Using the OARSI histopathology scoring system, cartilage damage and matrix proteoglycan depletionwere measured (Figure 5B). The average OARSI scores (0–24) were determined across treatment groups. As expected, a significant increase in OARSI score was found across DMM-operated mice in comparison to sham-operated animals (10.78 ± 1.97 vs. 2.44 ± 1.89). Pdgfrβ⁺ cell therapy led to the largest improvement in OARSI score (8.18 ± 2.35), while the Pdgfra⁺ cell therapy also showed a significant reduction in OARSI score in comparison to PBS control (9.02 ± 1.99). Results were stratified by gender, with similar findings (Figure S3).

3.5 Immunohistochemical staining of articular cartilage after Pdgfrα⁺ or Pdgfrβ⁺ cell therapy

Cartilage antigen expression was next assayed after Pdgfrα⁺ or Pdgfrβ⁺ cell therapy by immunohistochemistry, including aggrecan (ACAN), and type X collagen (ColX) (Figure 6). As expected, DMM induced a significant loss of ACAN immunoreactivity among superficial articular cartilage in comparison to sham-operated conditions (Figure 6A, upper rows). DMM-operated animals treated with either Pdgfrα⁺ or Pdgfrβ⁺ cell therapy showed partial restoration of Acan immunoreactivity (Figure 6A, lower rows).

Conversely, the expression of ColX, a marker of chondrocyte hypertrophy and severity of OA, showed the opposite expression patterns (Figure 6B). Here, and as expected, DMM induced a significant increase in ColX immunoreactivity (Figure 6B, upper rows). Both Pdgfrα⁺ and Pdgfrβ⁺ cell therapy reversed this increase in ColX expression among DMM-operated stifle joints (Figure 6B, lower rows).