Synovial fluid hyaluronan mediates MSC attachment to cartilage, a potential novel mechanism contributing to cartilage repair in osteoarthritis using knee joint distraction

Abstract

Objectives: Knee joint distraction (KJD) is a novel, but poorly understood, treatment for osteoarthritis (OA) associated with remarkable 'spontaneous' cartilage repair in which resident synovial fluid (SF) multipotential mesenchymal stromal cells (MSCs) may play a role. We hypothesised that SF hyaluronic acid (HA) inhibited the initial interaction between MSCs and cartilage, a key first step to integration, and postulate that KJD environment favoured MSC/cartilage interactions.

Methods: Attachment of dual-labelled SF-MSCs were assessed in a novel in vitro human cartilage model using OA and rheumatoid arthritic (RA) SF. SF was digested with hyaluronidase (hyase) and its effect on adhesion was observed using confocal microscopy. MRI and microscopy were used to image autologous dual-labelled MSCs in an in vivo canine model of KJD. SF-HA was investigated using gel electrophoresis and densitometry.

Results: Osteoarthritic-synovial fluid (OA-SF) and purified high molecular weight (MW) HA inhibited SF-MSC adhesion to plastic, while hyase treatment of OA-SF but not RA-SF significantly increased MSC adhesion to cartilage (3.7-fold, p<0.05) These differences were linked to the SF mediated HA-coat which was larger in OA-SF than in RA-SF. OA-SF contained >9 MDa HA and this correlated with increases in adhesion (r=0.880). In the canine KJD model, MSC adhesion to cartilage was evident and also dependent on HA MW.

Conclusions: These findings highlight an unappreciated role of SF-HA on MSC interactions and provide proof of concept that endogenous SF-MSCs are capable of adhering to cartilage in a favourable biochemical and biomechanical environment in OA distracted joints, offering novel one-stage strategies towards joint repair.

Keywords: Knee Osteoarthritis; Orthopedic Surgery; Osteoarthritis; Synovial fluid.

Figure 1

Antiadhesive nature of osteoarthritic (OA) synovial fluid (SF) on tissue culture plastic. (A) Direct plating of SF or replacement of SF by culture medium illustrating the extent to which synovial fluid-mesenchymal stromal cell (SF-MSC) adhesion is affected by osteoarthritic-synovial fluid (OA-SF). Gross (left) and magnified (right) images of culture morphology. (B) Differential adhesion of expanded SF-MSCs in OA-SF and rheumatoid arthritic-synovial fluid (RA-SF) (n=5 each, non-paired analysis). (C) Increase in adhesion of SF-MSCs in OA-SF after predigestion with hyase (n=5, paired analysis). (D) Differential effect of hyase treatment on SF-MSC adhesion in RA-SF and OA-SF (n=5 each, non-paired analysis). (E) Increasing concentrations of high, but not low molecular weight hyaluronan (HMWHA and LMWHA, respectively) inhibit the adhesion of SF-MSCs from three donors. *p<0.05, **p<0.001; individual samples are given in (C), median values with 25% (box) and 75% (whiskers) CI are presented in (B) and (D).

Figure 2

Osteoarthritic (OA) environment limits mesenchymal stromal cell (MSC) attached to cartilage surface. (A) Experimental setup of our novel in vitro adhesion assay. (B) Agarose gel electrophoresis illustrating inhibition of hyase by heparin. Heparin only, −/+; hyase only, +/−; heparin and hyase, +/+. (C) Representative confocal images showing a topographical view of adherent fluorescent micro-sized particles of iron oxide (FMPIO) labelled synovial fluid-mesenchymal stromal cells (SF-MSCs) (green) to superficial cartilage surface (grey), in the presence of culture medium (top), osteoarthritic-synovial fluid (OA-SF) (middle) and after pre-hyase treatment of OA-SF (bottom). (D) Quantification of labelled-MSC adhesion to cartilage surface relative to adhesion in culture medium, showing the consistent increase in adhesion upon pre-hyase digestion of OA-SF (n=5, paired analysis). (E) Equivalent data as in (D) showing SF-MSC adhesion to cartilage in rheumatoid arthritic-synovial fluid (RA-SF) (±hyase pretreatment). (F) Relative differences in adhesion expressed as a fold change of labelled MSCs adhered to cartilage surface after hyase treatment of RA-SF and OA-SF (n=5 each, non-paired analysis). *p<0.05, **p<0.001; individual samples are given in (D) and (E), median values with 25% (box) and 75% (whiskers) CI are presented in (F) and (D).

Figure 3

Synovial fluid promotes pericellular hyaluronan-coat formation and is responsible for differences in adhesion to cartilage. (A) Red blood cell (RBC) exclusion assay showing synovial fluid-mesenchymal stromal cells (SF-MSCs) without a hyaluronic acid (HA)-coat in culture medium (left, magenta arrow). SF-MSCs and the presence of an HA-coat after exposure to 10% OA-SF (middle, yellow arrow). Predigestion of OA-SF with hyase fails to induce formation of the HA-coat (right, blue arrow). (B) Highlighted cells from (A), with perimeter of the cell indicated by the coloured line. (C) RBC exclusion assay using rheumatoid arthritic (RA)-derived SF without hyase digestion (left); after prior digestion with hyase (right). (D) Confocal microscopy images of SF-MSCs exposed to osteoarthritic-synovial fluid (OA-SF) (top) and hyase treated OA-SF (bottom) confirming the presence and absence of the HA-coat under these conditions. SF-MSCs are stained green and RBCs stained red. (E) Quantification of the HA-coat area formed by native OA-SF and rheumatoid arthritic-synovial fluid (RA-SF) (n=7 and n=6, respectively; 109 measurements, 40 from OA and 69 from RA donors, non-paired analysis). (F) Representative gel electrophoresis and associated densitometry plot for OA-SF with and without hyase digestion. (G) Representative gel electrophoresis and corresponding densitometry plots showing example OA-SF compared with RA-SF. (H) Relative intensity of very high molecular weight HA (VHMWHA) (>9 MDa HA, represented by the grey dotted line in (F) and (G)) between native OA-SF and RA-SFs used in our in vitro adhesion assay (n=5 each by non-paired analysis). (I) Correlation between proportion of VHMWHA and fold change in adhesion, showing hyase treatment increases adhesion in those SF with more VHMWHA (r=0.88, p=0.002 n=9, non-parametric analysis). *p>0.05, **p<0.005.

Figure 4

In vivo osteoarthritis model and mesenchymal stromal cell (MSC) adhesion to cartilage under knee joint distraction. (A) Experimental setup of in vivo adhesion model; phases in the experiment are indicated in days. (B) Adipose tissue derived (AT)-MSC colony grown from canine fat pad digest. (C) Fluorescent micro-sized particles of iron oxide (FMPIO)-labelling of AT-MSCs (phase contrast, top; fluorescence, bottom). (D) Flow cytometry analysis of FMPIO uptake by AT-MSCs from each dog (green, red and blue histograms; grey histogram is non-labelled cells from a single dog). (E) Tri-lineage differentiation of AT-MSCs showing adiopgenesis (top and bottom), osteogenesis (AZ, alizarin red; ALP, alkaline phosphatase staining) and chondrogenesis (gross morphology, top; toluidine blue stained section, bottom). (F) Sagittal-MRI of a femur from a distracted limb showing presence of FMPIO-labelled MSCs (green arrows), a bone marrow lesion (yellow arrow) and cartilage defect (blue arrow). (G) Sagittal-MRI of a control joint without the addition of FMPIO-labelled AT-MSCs. (H) Topographical confocal images of cartilage defects adjacent to the bone marrow lesion in (F) showing the presence of FMPIO-labelled MSCs. (I) Topographical confocal image of control cartilage seen in (G). (J) Example gel electrophoresis and associated densitometry plot of synovial fluid (SF) from a matched contralateral control and distracted joint (>7 MDa, grey dotted line). (K) Relative proportion of high molecular weight hyaluronan (HMWHA) (>7 MDa) in each control and distracted joint. Matched animals are indicated by the black line. (L) Correlation between HMWHA in these joints and the percentage of total MSCs adhered per mm² of imaged joint surface. r=−0.6, p=0.2, n=6, non-parametric analysis.