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. 2025 Mar 19;17(3):e80844.
doi: 10.7759/cureus.80844. eCollection 2025 Mar.

Isolation and Analysis of Matched Osteoarthritic Cartilage Progenitor Cells and Bone Marrow Mesenchymal Stem Cells

Affiliations

Isolation and Analysis of Matched Osteoarthritic Cartilage Progenitor Cells and Bone Marrow Mesenchymal Stem Cells

Adam Esa et al. Cureus. .

Abstract

Introduction: Osteoarthritis (OA) is a chronic degenerative disorder that impacts synovial joints, leading to the degradation of articular cartilage and alterations in bone structure. As the most prevalent type of polyarthritis, its occurrence is increasing, particularly in Western countries. Current treatment options for OA involve various pharmacological therapies and prosthetic devices, which come with numerous limitations. Consequently, there is a growing interest among both patients and health care professionals in biological therapies, particularly the use of stem and progenitor cells for cartilage regeneration.

Methods: We extract articular cartilage progenitor cells (CPCs) and bone marrow mesenchymal stem cells (MSCs) from the femoral side of the knee joint of OA patients undergoing total knee arthroplasty. To isolate CPCs, digested full-depth chondrocytes from the femoral condyle undergo a fibronectin adhesion assay, while we separate bone marrow MSCs using the Ficoll™ density gradient centrifugation method. We expand both cell types in culture and measure their growth kinetics over 80 days. Additionally, we evaluate proliferation potential and senescence through bromodeoxyuridine incorporation and the senescence-associated β-galactosidase assay, respectively. Further, we analyze the expression of specific MSC markers in articular CPCs and bone marrow MSCs using flow cytometry. Results: We successfully isolated CPCs and bone marrow MSCs from matched osteoarthritic donors. The isolated CPCs and MSCs exhibit similar morphology and proliferation ability. Moreover, both cell types show positive expression for MSC markers CD-90, CD-105, and CD-166, while expressing low or no levels of CD-34 (a marker for hematopoietic stem cells) and exhibiting tri-lineage differentiation potential. Conclusion: We successfully isolate CPCs and bone marrow MSCs from the knee joints of osteoarthritic donors. Our findings indicate that both cell types demonstrate comparable morphology and growth kinetics, concurrently marking for classical MSC markers and exhibiting differentiation potential. These results are promising for the field of regenerative medicine. In this study, we outline the isolation of a rare group of matching mesenchymal stem/progenitor cells collected from the articular cartilage and bone marrow of patients undergoing total knee arthroplasty. This discovery lays the groundwork for comparative analyses, in that these cell types are primary candidates for cartilage-based regenerative therapies.

Keywords: articular cartilage; chondroprogenitor cells; osteoarthritis; regenerative medicine; stem cells.

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Conflict of interest statement

Human subjects: Consent for treatment and open access publication was obtained or waived by all participants in this study. South East Wales Research Ethics Committee issued approval 09/WSE04/35. Animal subjects: All authors have confirmed that this study did not involve animal subjects or tissue. Conflicts of interest: In compliance with the ICMJE uniform disclosure form, all authors declare the following: Payment/services info: All authors have declared that no financial support was received from any organization for the submitted work. Financial relationships: All authors have declared that they have no financial relationships at present or within the previous three years with any organizations that might have an interest in the submitted work. Other relationships: All authors have declared that there are no other relationships or activities that could appear to have influenced the submitted work.

Figures

Figure 1
Figure 1. Tibial plateau isolated from normal and osteoarthritic donors
The tibial plateau from a normal donor exhibits a smooth, intact articular cartilage surface with no visible abnormalities (A). In contrast, the osteoarthritic tibial plateau (B) displays characteristic features of OA, including cartilage erosion and exposure of the subchondral bone. OA, osteoarthritis
Figure 2
Figure 2. Phase contrast images of CPCs and MSC
(A) Colony-forming chondroprogenitor cells (CFU) from N-CPC. (B) OA-CPC. (C) Patient-matched BM-MSCs. (D) Isolated N-CPC after culture expansion. (E) OA-CPC after culture expansion. (F) Culture-expanded bone marrow MSCs. Scale bar: A-C = 100 µm. Scale bar: D-F = 200 µm. N-CPC, normal cartilage progenitor cell; BM-MSCs, bone marrow-derived mesenchymal stem cells; MSCs, mesenchymal stem cells; OA, osteoarthritis; CPC, cartilage progenitor cell
Figure 3
Figure 3. Initial adhesion to fibronectin and CFE
A and B display the initial percentage of chondrocytes adhering to fibronectin 24 hours after plating, with data from normal donors (n=5) and osteoarthritic donors (n=6), respectively. C and D illustrate the CFE at day 10 for CPCs isolated from normal (n=5) and OA donors (n=6). CPCs, cartilage progenitor cells; CFE, colony-forming efficiency; OA, osteoarthritis
Figure 4
Figure 4. BrdU (5-bromo-2-deoxyuridine) cell incorporation
The percentage of positively stained cells was calculated for each cell line (n = 3), with data presented as mean ± SEM. Blue bars represent OA-CPCs, red bars correspond to matched MSCs, and green bars indicate N-CPCs. N-CPC, normal cartilage progenitor cell; BM-MSCs, bone marrow-derived mesenchymal stem cells; OA, osteoarthritis; CPC, cartilage progenitor cell; BrdU, bromodeoxyuridine
Figure 5
Figure 5. Percentage of senescent cells in normal CPCs, OA-CPCs, and BM-MSCs
BM-MSCs, bone marrow-derived mesenchymal stem cells; OA, osteoarthritis; CPC, cartilage progenitor cell
Figure 6
Figure 6. BrdU labeling and senescence-associated β-galactosidase assay of CPCs and BM-MSCs
Representative images of CPCs isolated from normal (A, D) and osteoarthritic (B, E) cartilage, as well as BM-MSCs (C, F), stained for BrdU (A-C) and β-galactosidase (D-F). Scale bar = 200 µm. BM-MSCs, bone marrow-derived mesenchymal stem cells; OA, osteoarthritis; CPC, cartilage progenitor cell; N-CPC, normal cartilage progenitor cell; BrdU, bromodeoxyuridine
Figure 7
Figure 7. Percentage of B-galactosidase incorporation in matched CPC and MSCs
The percentage of β-galactosidase incorporation was measured in matched CPC lines derived from osteoarthritic cartilage (circles) and BM-MSCs (triangles) from five donors. Each cell line was analyzed in triplicate. Data are presented as mean values, and statistical differences were assessed using a Student’s t-test (P < 0.05). BM-MSCs, bone marrow-derived mesenchymal stem cells; CPC, cartilage progenitor cell; NS, not significant
Figure 8
Figure 8. FACS analysis of MSC surface markers
FACS analysis was conducted to assess the expression of CD-90, CD-105, CD-166, and CD-34 in CPC lines derived from both normal and osteoarthritic cartilage, as well as in OA-BM-MSCs. Representative histograms are shown for an OA-BM-MSC line (A-D), an OA-CPC line (E-H), and an N-CPC line (I-L). CD-34, a hematopoietic stem cell marker used as a negative control for MSCs, is displayed in D, H, and L for each respective cell line. The red-filled histogram represents positively labeled cells, while the unfilled histogram corresponds to the matched isotype control. FACS, fluorescence-activated cell sorting; OA-BM-MSCs, OA, osteoarthritis-matched bone marrow mesenchymal stem cells; BM-MSCs, bone marrow-derived mesenchymal stem cells; N-CPC, normal cartilage progenitor cell; OA, osteoarthritis; CPC, cartilage progenitor cell
Figure 9
Figure 9. Chondrogenic differentiation of isolated CPC and MSCs
A-C: Representative Toluidine blue-stained images of 3D pellet cultures from N-CPC (A), OA-CPC (B), and matched OA-BM-MSC (C) lines after 21 days of differentiation. D-E: Representative PCR analysis showing mRNA expression of aggrecan (ACAN, 100 bp), type II collagen (COL2A1, 165 bp), and GAPDH (positive control, 95 bp). Toluidine blue staining confirmed the presence of a glycosaminoglycan-rich matrix in all tested cell lines. Lane 1: N-CPC, Lane 2: OA-CPC, Lane 3: OA-BM-MSCs, Lane 4: NTC. Scale bars = 50 μm. NTC, no-template control; BM-MSCs, bone marrow-derived mesenchymal stem cells; N-CPC, normal cartilage progenitor cell; OA, osteoarthritis; CPC, cartilage progenitor cell
Figure 10
Figure 10. Osteogenic differentiation of isolated CPC and MSCs
A-C: Representative images of Alizarin Red staining in monolayer cultures of N-CPC, OA-CPC, and OA-BM-MSC lines after 14 days in an osteogenic medium. Alizarin Red staining was used to assess calcium deposition, a key marker of osteogenic differentiation. D-E: Representative PCR analysis showing mRNA expression of Runx-2 (164 bp), Osteonectin (143 bp), and GAPDH (control, 95 bp) in N-CPC, OA-CPC, and matched OA-BM-MSCs. All cell lines exhibited positive staining for Alizarin Red, indicating mineral deposition. Lane 1: N-CPC, Lane 2: OA-CPC, Lane 3: BM-MSCs, Lane 4: NTC. Scale bars = 50 μm. NTC, no-template control; BM-MSCs, bone marrow-derived mesenchymal stem cells; N-CPC, normal cartilage progenitor cell; OA, osteoarthritis; CPC, cartilage progenitor cell
Figure 11
Figure 11. Adipogenic differentiation of CPC and MSCs
A-C: Representative images of Oil Red O staining in monolayer cultures of N-CPC, OA-CPC, and OA-BM-MSC lines after one week in adipogenic medium. Lipid droplet accumulation is visible as red staining in all cell lines. D-E: Representative RT-PCR analysis of adipogenic differentiation markers, including PPAR-γ (78 bp) (D) and LPL (105 bp) (E), confirming adipogenic induction in monolayer cultures. GAPDH (95 bp) served as the housekeeping gene (E). Lane 1: N-CPC, Lane 2: OA-CPC, Lane 3: OA-BM-MSCs, Lane 4: NTC. Scale bars = 50 μm. PPAR-γ, peroxisome proliferator-activated receptor-γ; LPL, lipoprotein lipase; NTC, no-template control; BM-MSCs, bone marrow-derived mesenchymal stem cells; N-CPC, normal cartilage progenitor cell; OA, osteoarthritis; CPC, cartilage progenitor cell

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References

    1. Esa A. PhD Thesis, Cardiff University. Cardiff University; 2015. Characterising the Role of Articular Cartilage Progenitor Cells in Osteoarthritis.
    1. Wnt signaling and osteoarthritis. Luyten FP, Tylzanowski P, Lories RJ. Bone. 2009;44:522–527. - PubMed
    1. Stem cells and cartilage development: complexities of a simple tissue. Hollander AP, Dickinson SC, Kafienah W. http://10.1002/stem.534. Stem Cells. 2010;28:1992–1996. - PMC - PubMed
    1. Osteoarthritis: National Clinical Guideline for care and management in adults. Royal College of Physicians. [ Jan; 2025 ]. 2008. https://pubmed.ncbi.nlm.nih.gov/21290638/ https://pubmed.ncbi.nlm.nih.gov/21290638/ - PubMed
    1. National Clinical Guideline Centre (UK) Arthritis. Arthritis and Musculoskeletal Alliance. London, UK: National Institute for Health and Care Excellence; 2014. Osteoarthritis: Care and Management in Adults. - PubMed

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