A three-dimensional in vitro culture environment of a novel polymer scaffold, yielding chondroprogenitors and mesenchymal stem cells in human chondrocytes derived from osteoarthritis-affected cartilage tissue

Abstract

Objective

We evaluated the expression of stem/progenitor biomarkers in osteoarthritic tissue derived chondrocytes cultured using a three-dimensional (3D) thermo-reversible gelation polymer (TGP).

Methods

The chondrocytes from discarded biopsy tissues obtained from human elderly patients with osteoarthritis were cultured using the 3D-TGP up to six weeks.

Results

The chondrocytes grew in a tissue-like manner, without de-differentiation into fibroblasts, and the cells thus tissue-engineered were proven positive for CD49e, OCT4, CD-105 and STRO-1 by immunohistochemistry.

Conclusion

This study establishes the efficacy of this 3D-TGP platform for clinically useable in-vitro tissue-engineered cartilage for improvising the clinical outcome of cell therapy for cartilage repair.

1. Introduction

Osteoarthritis (OA) is a highly prevalent debilitating disease that greatly affects quality of life in both the developing and developed worlds.¹ OA is characterized by articular cartilage degeneration, synovial inflammation and alterations in the adjoining soft tissues and the sub-chondral bone.¹ Attempts to repair the cartilage damage have given rise to several therapies, such as microfracture, osteo-chondral autografts and cell therapies, including autologous chondrocyte implantation (ACI) and scaffold-based matrix-associated chondrocyte implantation (MACI). Though no technique has, to date, been completely successful in regenerating the articular cartilage to its full efficiency and function, ACI and MACI have been described as potential therapies for regenerating the damaged cartilage.¹ A major limitation of ACI is the in vitro and in vivo de-differentiation of the hyaline chondrocytes to a fibroblast phenotype, leading to a less optimal clinical outcome. MACI offers a solution, but ACI and MACI generally utilize the normal non-weight bearing cartilage tissue to derive the chondrocytes, and chondrocytes from osteoarthritic (OA) cartilage have been considered as less appropriate for use, even after they have been proven to restore the hyaline phenotype after three-dimensional (3D) scaffold-based culture.² In our earlier studies, we proved the long-term hyaline phenotype maintenance of human chondrocytes, both in vitro and in vivo, when a thermo-reversible gelation polymer (TGP)-based 3D culture-platform was employed.^3,4 We also reported the higher expression of the pluripotent stem cell marker α1-2 Fuc recognition lectin, UEA-1 in lectin microarray in chondrocytes derived from such OA-affected cartilage tissue of elderly patients with severe osteoarthritis, tissue-engineered in the 3D-TGP for 18 weeks in vitro.⁵ As we wanted to explore the types of stem/progenitor cells that could explain the pluripotency marker's expression in the lectin microarray, we undertook the current study to characterize the long-term 3D-TGP tissue-engineered hyaline cartilage, using immunohistochemistry for established pluri-multipotent stem/progenitor cell surface markers.

2. Methods

The study was approved by the institutional ethics committee of Edogawa Hospital, Tokyo, Japan. Human chondrocytes were obtained from discarded cartilage biopsies of elderly patients (aged 60–85 years) undergoing arthroscopy. The study was conducted in accordance with relevant guidelines/regulations and informed consent was obtained from all participants and/or their legal guardians. Cartilage tissue obtained was processed using the methodology reported by us earlier.3, 4, 5 Briefly, the cartilage tissue was weighed and subjected to digestion with 0.25% trypsin for 30 min in an orbital shaker at 150 rpm at 37 °C. The tissues were then subjected to 2 mg/ml collagenase digestion for 12–18 h at 37 °C in an orbital shaker, followed by filtration with a 70-μm nylon mesh. After the undigested tissues were discarded, the filtrate was centrifuged at 1000 rpm for 10 min. The cells obtained were counted by the trypan blue dye exclusion method. After two-dimensional (2D) monolayer culture in media containing low glucose D-MEM, 10% autologous plasma, 1% penicillin streptomycin, 50 μg/ml Gentamicin, 0.25 μg/ml amphotericin B, and l-ascorbic acid (50 mg/ml) for 2 weeks at 37 °C with 5% CO2, the cells were seeded into the 3D-TGP scaffold using the same media composition and cultured for six weeks at 37 °C with 5% CO2 in an orbital shaker. The 2D cultured cells and the 3D-TGP cultured tissue, after the said period of culture, were fixed using formalin, and then embedded in paraffin blocks. Serial sections were deparaffinized and stained with hematoxylin and eosin (H&E), employing standard histological techniques. Deparaffinized 3D-TGP tissues were sectioned at 4 μm thickness and subjected to immunohistochemical staining for CD34 Antibody (Ventana 518-102418), Anti-Integrin alpha 5/CD49e Antibody (10F6, NBP2-37666), STRO-1 Antibody (NBP1-48356), SOX2 Antibody (4G8, NBP2-29623), Endoglin/CD105 Antibody (CL1912, NBP2-34493) and OCT4 Antibody (OTI9B7, NBP1-47923). Anti-CD34 antibody was purchased from Ventana, USA, and other antibodies were purchased from Novusbio, USA. Staining was performed using a Ventana Benchmark XT (Ventana Medical Systems) automated slide-staining system. The sections were deparaffinized, pre-treated with Cell Conditioning 1 (CC1, Ventana Medicals Systems), reacted with primary antibodies for 32 min at room temperature, visualized with an iView DAB Detection Kit (Ventana Medical Systems), and counter-stained with hematoxylin (Ventana Medical Systems) and Bluing Reagent (Ventana Medical Systems). Human tissues were used as positive controls for the immunostaining, for positive expression of the corresponding antibodies mentioned above (these tissues were used with informed consent from the patients based on agreements between the hospitals and the Tokyo Central Pathology Laboratory (TCPL), Japan) and negative controls were prepared by adding REAL Antibody Diluent (Dako), instead of the primary antibody.

3. Results and Discussion

The weight of the cartilage tissues obtained after biopsy ranged from 0.1 to 0.3 g. The average initial cell count was 0.43 × 10⁶ cells. The average cell count obtained after 2D monolayer culture after two weeks was 7.75 × 10⁶ cells, but fibroblast de-differentiation phenotype was observed (Fig. 1 A and C). After seeding in 3D-TGP, the chondrocytes grew as a tissue-like structure (demonstrating re-differentiation to hyaline phenotype) without de-generation up to six weeks, which was observed both during in vitro culture and in H & E staining (Fig. 1 B and D). Immuno-histochemistry showed positivity for CD49e, OCT4, CD-105 and STRO-1 (Fig. 2) and negativity for CD-34 for the tissues grown in 3D-TGP. CD49e has been reported by Vinod et al.⁶ as a distinguishing marker for chondroprogenitors derived from human cartilage. The minimal criteria of the International Society for Cellular Therapy (ISCT) for defining multipotent mesenchymal stromal cells includes positive expression of CD-105 and lack of expression of CD-34.⁷ For more than 25 years, the STRO-1 antibody has been defined as a hallmark of immature pluripotent mesenchymal precursor cells (MPC) and is a widely used marker for mesenchymal stem cells (MSC).^8,9 In fact, high levels of STRO‐1 antigen expression have been associated with an immature MSC phenotype, which has high proliferative and multidifferentiation potential.⁹ OCT4 is an established marker of a cell with a pluripotent embryonic stem cell identity and it is a key regulator of pluripotent cells across mammalian species.¹⁰ In this study, we have been able to tissue-engineer hyaline chondrocytes derived from inflamed OA tissues into clinically useable cartilage constructs using the 3D-TGP platform, which contains sub-populations that have stem cell (mesenchymal and pluripotent) and progenitor cell properties. Stem and progenitor cell properties further enhance the regenerative capabilities of such tissue constructs for use in cartilage-tissue engineering. 3D-TGP not only restores the native cartilage phenotype, but also helps to provide an environment that nurtures stem and progenitor cell populations to thrive in it.

Fig. 1.

A. Cells de-differentiated into fibroblasts during monolayer 2D culture for 2 weeks; B. Cells growing as a tissue-like construct in 3D-TGP up to 6 weeks; C. H & E staining of monolayer 2D cultured cells (individual cells observed); D. H & E staining of monolayer 3D-TGP culture exhibiting continuous tissue-like morphology.

Fig. 2.

Immuno-histochemistry staining showing A–E: Positive Controls (A: CD-34; B: CD49e; C: CD-105; D: OCT4; E: STRO-1). F–J: Staining of study samples (3D-TGP tissue engineered cartilage constructs) - F. Negative staining for CD-34; G. Positive staining for CD49e; H. Positive staining for CD-105; I. Positive staining for OCT4 and J. Positive staining for STRO-1.

4. Conclusion

Osteoarthritic chondrocytes obtained from elderly patients can be rejuvenated to form native hyaline phenotype cartilage tissue in vitro in a 3D environment of thermo-reversible gelation polymer (TGP), which helps in enhanced expression of markers pertaining to mesenchymal stem cells, chondroprogenitors and cells of pluripotent lineage up to 6 weeks. These potentials of the 3D-TGP environment may therefore be considered for use as a matrix for encapsulation while transplanting in vitro-expanded human chondrocytes to address cartilage damage after necessary clinical validation.

Funding

No funding was received for conducting this study.

Informed consent

Informed consent was obtained from all participants and/or their legal guardians whose data has been included in the study.

Ethical approval

The institutional ethics committee of Edogawa Hospital, Tokyo, Japan approved the study. All procedures performed were in accordance with the ethical standards of the institutional ethics committee of Edogawa Hospital, Tokyo, Japan and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards.

Declaration of competing interest

• 1.Author Katoh is an employee of Edogawa Hospital, Japan and is an applicant/inventor to several patents on biomaterials and cell culture methodologies, some of them described in this manuscript.
• 2.Author Yoshioka is an employee of Mebiol Inc and an applicant to several patents on TGP and its applications
• 3.Author Abraham is a shareholder in GN Corporation Co. Ltd., Japan and is an applicant/inventor to several patents on biomaterials and cell culture methodologies, some of them described in this manuscript.