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. 2020 Mar 2;43(1):e20190275.
doi: 10.1590/1678-4685-GMB-2019-0275. eCollection 2020.

Combining canine mesenchymal stromal cells and hyaluronic acid for cartilage repair

Maria Inês Wits  1 , Gabriela Cabanas Tobin  2 , Maiele Dornelles Silveira  2   3 , Karine Gehlen Baja  1 , Luisa Maria Macedo Braga  3 , Patricia Sesterheim  4 , Melissa Camassola  2 , Nance Beyer Nardi  2   3   4
Affiliations

Combining canine mesenchymal stromal cells and hyaluronic acid for cartilage repair

Maria Inês Wits et al. Genet Mol Biol. .

Abstract

Cell therapy and tissue engineering have been intensively researched for repair of articular cartilage. In this study, we investigated the chondrogenic potential of canine adipose-derived mesenchymal stromal cells (ASCs) combined to high molecular weight hyaluronic acid (HA) in vitro, and their therapeutic effect in dogs with chronic osteoarthritis (OA) associated with bilateral hip dysplasia. Canine ASCs were characterized after conventional 2D culture or 3D culture in HA, showing adequate immunophenotype, proliferation and trilineage differentiation, as well as chondrogenesis after cultivation in HA. ASC/HA constructs were used to treat 12 dogs with OA, sequentially assigned to control, ASC and ASC/HA groups. Animals were examined for clinical, orthopedic and radiological parameters. Lameness at walk and pain on manipulation were reduced in the ASC group and mainly in the ASC/HA group. Range of motion and detection of crepitus on hip rotation and abduction improved similarly in all groups. For articular edema, muscle atrophy, Norberg angle values and radiographic analyses, there were no variations throughout the period. These results indicate that ASC/HA constructs are safe and may be an effective therapeutic tool in treating canine chronic osteoarthritis, which should be confirmed with larger studies and additional clinical parameters.

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Figures

Figure 1
Figure 1. (A) Representative photographs showing the trilineage differentiation capacity of canine ASCs; control cultures were maintained in normal medium. Scale bars, 100 μm. (B) ASC proliferation rate along 60 days of culture. (C) Immunophenotype of canine ASCs (n = 1, analyzed in triplicate). Representative results show that cultures were negative for CD45 and MHC class II, and positive for CD90.
Figure 2
Figure 2. Proliferation rate of canine ASCs cultured for 3 days in normal 2D conditions (Control) or encapsulated in HA, assessed by DNA assay (n = 3).
Figure 3
Figure 3. Phase contrast aspect of ASCs encapsulated in HA and cultured for 7, 21 or 28 days in normal medium (A, B, C) or chondrogenic-inducing medium (D, E, F). Scale bars = 100 μm.
Figure 4
Figure 4. Chondrogenic matrix stained with Alcian blue in encapsulated ASC/HA constructs cultured for 28 days in normal (A) or chondrogenic-inducing (B) medium. A weak background staining can be seen in HA alone (C). (D) Detail of a more dense chondrogenic structure which can also be observed in the constructs. Original magnification x200 (A and B) or x100 (C and D).
Figure 5
Figure 5. GAG (μg) quantification normalized to protein concentration (mg) of ASC-HA constructs cultured for 28 days (n=3). **p < 0.01.
Figure 6
Figure 6. Representative cross-section images of encapsulated ASC/HA constructs cultured for 28 days with chondrogenic-inducing medium, stained with Alcian blue. (A , B, C) Different aspects of the chondrogenic matrix. (D) Control (Synvisc® alone). Original magnification x100.
Figure 7
Figure 7. Results of baseline (day 0) or follow-up (days 30, 60 and 90) evaluations of patients in groups control (treated with PBS), ASC (treated with adipose tissue-derived mesenchymal stromal cells) and ASC/HA (treated with ASCs + hyaluronic acid). * p < 0.05; ** p < 0.001, ANOVA and Tukey’s post hoc test.
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