Intra-articularly injected mesenchymal stem cells promote cartilage regeneration, but do not permanently engraft in distant organs

Viability of MSCs after injection

In order to assess the effects of injection on cell viability, we tested MSC viability over 5 days after ejection through a 23 G needle in vitro. MSCs were maintained in culture, and LDH and PrestoBlue analyses were performed. Measurement of LDH activity, an index of cytotoxicity, showed no differences between the ejected and non-ejected control MSCs (Fig. 1A). Similarly, no significant differences between the two groups were observed in the metabolic activity at the different time points (Fig. 2B). However, the metabolic activity in both control and ejected MSCs increased over time, suggesting that the injection procedure did not change the cells’ proliferating behavior.

MSCs distribution and engraftment in the knee joint

To analyze distribution and engraftment of intra-articularly injected MSC, we used a full thickness articular cartilage defect model in the patellofemoral groove in the current study. This type of defect exposes the subchondral bone, and leads to the initial formation of a fibrin clot at the injury site. The knee joints from the recipient rats (ALPP^m) were dissected and histochemically analyzed to study the presence of ALPP-labeled MSCs over time (Fig. 2). We did not observe any adverse effects after intra-articular injection of MSCs or serum. There were no animals excluded from the analyses. As expected, a large number of donor MSCs was observed in the knee joints, 1 day after cell delivery. Furthermore, cells were not homogenously distributed through the knee cavity, but formed cell aggregates suspended in the synovial fluid. A few cells were observed in the defect area (3.1 ± 1.3%), suggesting their intrinsic capacity to migrate rapidly towards the injury site. Nevertheless, most cells remained in the synovial cavity (16.9 ± 3.5%) and some cells were found in the suprapatellar bursa space (2.6 ± 0.9%). One week and 1 month after cell injection, ALPP-labeled MSCs were also detected in the cartilage lesion (2.3% ± 0.8 and 5.1% ± 1.6, respectively) and the synovial cavity (1.5% ± 0.4 and 1.2% ± 0.5, respectively). However, the number of ALPP-labeled MSCs in these localizations was decreasing over time. Similarly, the number of ALPP-labeled MSCs in the suprapatellar bursa also declined with time (1.8% ± 0.8 at 1 week post-injection), and no cells were observed at and beyond 1 month after cell delivery (Fig. 2B). Two and 6 months after MSC injection, ALPP-labeled MSCs were neither present in the cartilage lesion area nor in the knee cavity (Fig. 2A)

Migration of MSCs to distant organs

Different distant organs were collected from the transgenic recipient rats that were intra-articularly injected with donor ALPP-labeled MSCs. Histochemical staining of the different distant organs revealed the presence of ALPP-labeled MSCs in the perivascular space around pulmonary blood vessels in one of the studied animals, 1 day after cell injection (Fig. 3). However, no donor transgenic MSCs were detected in the lung of the recipient rats at later time points (1 week, 1 month, 2 months or 6 months after cell injection). Furthermore, we failed to identify ALPP-labeled MSCs in the heart, spleen, kidney or liver at 1 day, 1 week, 1 month, 2 months and 6 months after cell delivery.

Therapeutic efficacy of MSCs for cartilage regeneration

To evaluate cartilage regeneration, toluidine blue and safranin O stainings were performed. Representative photomicrographs of the focal articular cartilage defects at 1 month, 2 months, and 6 months after ALPP-labeled MSC injection are shown in Fig. 4. One month after cell injection, tissue similar to neocartilage was observed in the MSC-injected group, whereas unsatisfactory tissue integration and apparent tissue gaps were found in the control and serum groups. Compared with control and serum-injected rats, which showed poor matrix staining as well as abnormal tissue structure, improved matrix staining was observed in the defects, 2 months after MSC injection. Notably, MSCs-treated rats exhibited almost normal hyaline cartilage tissue at the defect site, 6 months after cell injection. Toluidine blue and safranin O staining within the defect site was similar to the surrounding tissue, suggesting the formation of healthy neocartilage. In contrast, both stainings revealed a poor content of proteoglycans in the neotissue matrix in control and serum samples.

Further evaluation of the collagen content at the defect sites was performed by immunofluorescence staining (Fig. 5). Collagen type I (COL1) was highly expressed in the cartilage defects of control and serum-injected rats. Conversely, a lower expression of COL1 was found in defects of MSC-treated animals. Interestingly, collagen type X (COL10), a marker of hypertrophic cartilage, was also highly expressed in the defects in control and serum-injected knees, but was almost undetectable in defects of MSC-injected rats. In contrast, defects from MSC-injected rats showed a higher abundance of collagen type II (COL2), the main component of healthy articular cartilage.

Evaluation of the inflammatory response after IA injection of MSCs

In order to investigate the modulatory effect of MSCs on the inflammatory response resulting from the surgical procedures and the cartilage lesion, we analysed the presence of CD68⁺macrophages and of interleukin 1β (IL1β) by immunofluorescence, 1 day after MSC injection (Fig. 6A). We observed macrophages and IL1β expression in the suprapatellar bursa, synovial tissue and at the defect site. Interestingly, ALPP-labeled MSCs were also located in these regions, and were surrounding CD68- and IL1β-positive stained areas. The IL1β protein expression in the cartilage defects tended to be higher in control and serum-injected rats, compared with MSC-treated animals. However, the differences between the groups were not statistically significant due to a large inter-individual variance (Fig. 6B).

Cytotoxicity and metabolic activity assays

MSCs were diluted to a final cell density of 10⁷ MSCs in 50 µL of native rat serum. A 1 mL syringe with a 23 G needle was used to deliver cells in a slow and steady injection into cell cultures dishes, mimicking the injections that were performed for intra-articular delivery of MSC in vivo. Post-ejection MSCs and non-ejected control MSCs were maintained in cell culture medium and incubated under hypoxic conditions. Analyses were performed 1 hour after ejection and at later time points (1, 2 and 5 days after ejection). The presence of lactate dehydrogenase (LDH) in culture media was used as an index of cell death. LDH activity was determined following the manufacturer’s instructions (Pierce LDH Cytotoxicity Assay Kit, Thermo Scientific, Rockford, USA). Cell metabolism was measured using PrestoBlue (Invitrogen, Carlsbad, CA, USA), following the manufacturer’s protocol. Data was normalized to the control group (100%).

Animal model

The animal model used in this study is based on the human placental alkaline phosphatase, ALPP, as cell tracking marker. This enzyme is heat stable, but can be transformed into a heat-sensitive form by changing only one amino acid. Two different transgenic rat lines on inbred F344 genetic background were used for the experiment: a donor line expressing the heat-resistant protein (ALPP) and a recipient line expressing the heat-sensitive protein (ALPP^m). Because these two protein forms differ in only a single amino acid, ALPP^m-expressing recipient rats are tolerant to ALPP-labeled cells and tissues isolated from the transgenic ALPP donors²⁸. All animals were housed in individually ventilated cages (Techniplast) at 24 °C and a 12 h/12 h light/dark cycle with free access to a standard rat chow and tap water. Two male rats were housed per cage.

Focal cartilage defect

A focal full thickness articular cartilage defect was surgically created in the immunocompetent transgenic ALPP^m recipient rats. Briefly, cartilage defects were placed using a 0.7-mm NIH Style Neuro Punch (Fine Science Tools [FST]) in the patellofemoral groove of the left knee of male recipient animals at the age of 4–6 months under isoflurane anesthesia. The joint capsule and muscle tissue were sutured using 6-0 Vicryl (Ethicon), and the skin was closed using 4-0 Vicryl. For analgesia, oral metamizole (Novalgin™: 50 µL) was administered 30 min before and 6 h after surgery. Antibiosis was performed over 5 days after surgery using enrofloxacin (Baytril: 10 mg/kg s.c. once daily). The anti-inflammatory drug carprofen (Rimadyl: 20 µL s.c. once daily) was administered over 3 days after surgery in order to manage pain.

Isolation and culture of BM-MSCs

Long bones including femora, tibiae and humeri were collected from 4- to 6-week-old ALPP-transgenic male rats for isolating bone marrow-derived mesenchymal stem cells (BM-MSCs). Animals were sacrificed by CO₂ immediately before bone collection. Briefly, bones were cut at both epiphyses and incubated in cell culture medium containing collagenase. The bone marrow was flushed out, and Ficoll gradient centrifugation was performed for collecting the mononuclear cell fraction. Non-adherent cells were removed and MSCs were cultured under hypoxic conditions (37 °C, 5% CO₂, and 5% O₂) up to passage 5 as described previously⁵⁴. Using this protocol, we reported in detail earlier that bone marrow-derived MSCs isolated from ALPP-transgenic donor rats maintained their immune phenotype as well as their differentiation capacity over six passages during in vitro cultivation⁵⁴.

IA injection of ALPP-labeled MSCs

For IA injection, 10⁷ transgenic donor MSCs were suspended in 50 µL of WT native rat serum isolated from whole blood. MSCs were intra-articularly injected using a 23 G needle into the knee cavity of transgenic ALPP^m recipients under isofluorane anesthesia, 2 weeks after defect creation. The knee joint was kept in a bent position, patella and femur were palpated, and ALPP-labeled MSCs were intra-articularly injected at a 90° angle from the medial side in the space between femur, tibia, and patella. Control animals were injected with rat serum, or left untreated. The rats (n = 4 per group and time point) were randomly allocated to the three groups, i.e., MSC-treated, serum control, and untreated. The body weight of the rats allocated to the treatment groups did not differ prior to treatment. For all experiments, the experimental unit was a single animal.

Histological assessment

Heart, spleen, kidney, liver, and lung from ALPP^m recipient rats were collected 1 day, 1 week, 1 month, 2 months, and 6 months after injection of ALPP-labeled MSCs. Tissues were fixed in 40% ethanol and embedded in paraffin. Five-μm-thick sections on 3-aminopropyltriethoxy-silane (APES, Sigma) pretreated slides were prepared and 30–50 tissue sections per animal and organ were randomly selected from different tissue regions for further staining and evaluation. Briefly, samples were rehydrated, and heated at 72 °C for 1 h to block both endogenous ALP and ALPP^m activity. Sections were then incubated in TNM buffer (0.1 M Tris-HCl, pH 9.5, 0.1 M NaCl, 5 mM MgCl₂) containing 0.17 μg/ml of the substrate 5-bromo-4-chloro-3-indolyl phosphate (BCIP; Sigma) and 0.45 μg/ml nitrotetrazolium blue chloride (NBT; Sigma) at room temperature overnight to detect ALPP activity. Subsequently, sections were counterstained with methyl green (Vector), dehydrated, and mounted using Vectamount (Vector).

Undecalcified dissected knee joints were directly embedded in frozen section compound (FSC 22; Leica) and snap-frozen in liquid nitrogen. Frozen samples were sectioned at 7 μm thickness on a Leica CM1950 cryostat with the help of an adhesive tape⁵⁵. BCIP/NBT staining was performed as described above but reducing the heating time to 10 min in order to preserve tissue integrity in the cryosections. ALPP-positive area was quantified using ImageJ (NIH). Three different regions were identified and evaluated: synovial cavity, suprapatellar bursa and defect area. The total area as well as the corresponding ALPP-stained area in each region were measured and the presence of ALPP-labeled MSCs was given as percentage of total area. The defect site in every tissue section was identified with the help of the neotissue formed in the damaged area, as it is not exactly equal to the surrounding healthy cartilage. Knee joint cryosections were stained with toluidine blue (Sigma) and safranin O (Sigma) to evaluate the cartilaginous matrix. All histological analyses were performed in a non-blinded fashion.

Immunofluorescence analysis

Knee cryosections were fixed with ice-cold ethanol and blocked with blocking buffer for 1 h at room temperature. The cryosections were incubated with primary antibodies against type I collagen (600-401-103, Rockland), type II collagen (CP18, Millipore), type X collagen (ab58632, Abcam), interleukin-1 beta (ab9722, Abcam), CD68 (MCA341R, BioRad), and ALPP (GTX72989, GeneTex or CBL207, Chemicon). Thereafter, sections were incubated with a mixture of FITC-conjugated anti-mouse (F0382, Sigma) and Alexa Fluor 555–conjugated anti-rabbit (4413, Cell Signaling) antibodies for 1 h at room temperature, air-dried, and mounted with Fluoromount (21644.01, Serva). DAPI (4083 S, Cell Signaling) was used to visualize nuclei. All immunofluorescence analyses were performed in a non-blinded fashion. Collagen stainings within the defect region were quantified as percent of defect area, using ImageJ.

Statistical analysis

All data are presented as mean values ± standard error of the mean (SEM). Normal distribution of data was analyzed by Kolmogorov-Smirnov test. Differences between two independent groups were assessed using 2-tailed Student’s t-test. Data from more than two groups were analyzed using one-way analysis of variance (ANOVA) followed by Tukey’s multiple comparison test, or Kruskal-Wallis test followed by Mann-Whitney U-test, as appropriate (SPSS, Chicago, IL, USA). P < 0.05 was considered statistically significant.

Study approval

All animal studies were approved by the Ethical Committees of the University of Veterinary Medicine, Vienna, and of the Austrian Federal Ministry of Science and Research, (permit no. BMWF-68.205/0146-II/3b/2013) and were undertaken in strict accordance with prevailing guidelines for animal care and welfare.