Device-Based Enrichment of Knee Joint Synovial Cells to Drive MSC Chondrogenesis Without Prior Culture Expansion In Vitro: A Step Closer to 1-Stage Orthopaedic Procedures

Abstract

Background: Synovial fluid (SF) mesenchymal stem cells (MSCs) are derived from the synovial membrane and have cartilage repair potential. Their current use in clinical practice is largely exploratory. As their numbers tend to be small, therapeutic procedures using MSCs typically require culture expansion. Previous reports indicate that the stem cell-mobilizing device (STEM device) intraoperatively increases SF-MSCs.

Purpose: This study evaluated the chondrogenic potential of non-culture expanded synovium-mobilized MSCs and SF-microfragments obtained after enrichment using the STEM device and ascertained if device-mediated synovial membrane manipulation facilitated ongoing MSC release.

Study design: Controlled laboratory study.

Methods: Two samples of aspiration fluid were collected intraoperatively before and after STEM device utilization from patients (n = 16) undergoing diagnostic or therapeutic knee arthroscopy. Human knee synovium (n = 5) was collected during total knee replacement, and a suspended culture was performed to assess the effect of the STEM device on ongoing MSC release. Colony forming unit-fibroblastic assays were used to determine the number of MSCs. Additionally, cytometric characterization of stromal and immune cells and chondrogenesis differentiation assay were performed without culture expansion. Filtered platelet concentrates were prepared using the HemaTrate system.

Results: After STEM device use, a significant increase was evident in SF-MSCs (P = .03) and synovial fluid-resident synovial tissue microfragments (P = .03). In vitro-suspended synovium released significantly more MSCs following STEM device use than nonstimulated synovium (P = .01). The STEM device-released total cellular fraction produced greater in vitro chondrogenesis with significantly more glycosaminoglycans (GAGs; P < .0001) when compared with non-STEM device synovial fluid material. Nonexpanded SF-MSCs and SF-microfragments combined with autologous filtered platelet concentrate produced significantly more GAGs than the complete chondrogenic media (P < .0001). The STEM device-mobilized cells contained more M2 macrophage cells and fewer M1 cells.

Conclusion: Non-culture expanded SF-MSCs and SF-microfragments had the potential to undergo chondrogenesis without culture expansion, which can be augmented using the STEM device with increased MSC release from manipulated synovium for several days. Although preliminary, these findings offer proof of concept toward manipulation of the knee joint environment to facilitate endogenous repair responses.

Clinical relevance: Although numbers were small, this study highlights 3 factors relevant to 1-stage joint repair using the STEM device: increased SF-MSCs and SF-microfragments and prolonged synovial release of MSCs. Joint repair strategies involving endogenous MSCs for cartilage repair without the need for culture expansion in a 1-stage procedure may be possible.

Keywords: nonexpanded cells; platelet products; synovial resident mesenchymal stem cells.

Figure 1.

Enumeration of resistant SF-MSCs, Sm-MSCs, and SF-microfragments. (A) CFU-F numbers in aspiration and mobilized samples containing SF-MSCs and Sm-MSCs, respectively (n = 6). (B) Colonies derived from SF-MSCs and SF-microfragments. (C) SF-microfragment numbers before and after mobilization (n = 6). (D) Hematoxylin and eosin–stained SF-microfragments after mobilization. Scale bar = 100 µm. Values are presented as mean ± SD. *P < .05. CFU-F, colony-forming unit–fibroblastic; MSC, mesenchymal stem cell; SF, synovial fluid; Sm, synovial mobilized.

Figure 2.

Chondrogenic potential of SF-MSCs and Sm-MSCs. (A) The number of platelets, white blood cells (WBCs), and red blood cells (RBCs) before and after filtration (n = 11). (B) Production of GAGs by culture-expanded SF-MSCs after 21 days with CCM, 50% AUT-fPC, 50% ALL-fPC, and negative control (DMEM media only) (n = 5) with representative images of toluidine blue–stained sections of SF-MSC pellets. Scale bar = 0.5 mm. (C) Expression of the chondrogenic markers in SF-MSC pellets (n = 3). (D) GAG production by SF-MSCs (from initial aspiration) after 21-day culture with CCM, AUT-fPC, and negative control (DMEM media only) (n = 5) with representative images of toluidine blue–stained sections of SF-MSC pellets. Scale bar = 0.5 mm. (E) Comparison of SF-MSC and Sm-MSC GAG production and representative images of toluidine blue–stained sections after 21-day culture with CCM (n = 6). Scale bar = 0.5 mm. (F, G) Expression of chondrogenic, anabolic, and catabolic markers (n = 3). Values are presented as mean ± SD. ns, not significant. *P < .05. **P < .01. ***P < .001. ****P < .0001. ALL, allogenic; AUT, autologous; CCM, complete chondrogenic differentiation medium; ΔCT, the difference between gene Ct and house keeping gene CT; DMEM, Dulbecco’s modified Eagle medium; fPC, filtered platelet concentrate; GAG, glycosaminoglycan; MSC, mesenchymal stem cell; SF, synovial fluid; Sm, synovial mobilized.

Figure 3.

Surface marker expression of initial aspiration and mobilized joint aspirates: a comparison of relative frequencies of CD90^HighCD45^Low and macrophage subpopulations in nonexpanded initial aspiration and mobilized joint aspirates (n = 6). ns, not significant. *P < .05. HLA-DR, human leukocyte antigen-DR; MSC, mesenchymal stem cell; SF, synovial fluid; Sm, synovial mobilized.

Figure 4.

Effect of agitation on Sm-MSC release from the synovium. (A) Synovium from total knee replacement was divided into 2 parts with identical weight. (B) Methylene blue staining of colonies produced from released cells in suspended cultures. (C) Comparison of CFU-F numbers for suspended cultures for 4, 8, and 12 days of culture (n = 5). Values are presented as mean ± SD. *P < .05. CFU-F, colony forming unit–fibroblastic; ns, not significant.