Co-culture of human chondrogenic microtissues with osteoblast-like Saos-2 cells or HFF-1 fibroblasts influence the differentiation potential of spheroids

Abstract

BACKGROUND:

Tissue engineering has become a major field of research in biotechnology and biomedicine. As a consequence, cell-based therapeutic approaches are entering the hospitals, especially for skeletal regeneration. Traumatic injuries of cartilage are treated with autologous cell suspensions or in vitro generated cartilage tissues, but there is actually no therapy available for degenerative cartilage defects. However, Osteoarthritis (OA) is a major public health problem in the world affecting 240 million people globally.

OBJECTIVE:

To develop suitable in vitro tissues, the properties of chondrogenic spheroids should be optimized via co-culture with cells naturally occurring as joint neighbours.

METHODS:

Human chondrocytes were isolated from condyles and propagated in monolayer culture. Scaffold-free spheroids were generated and co-cultured with joint-specific partner cells (osteoblast-like osteosarcoma cells, fibroblasts). Morphology and differentiation was analyzed using histochemistry (Alcian blue, Safranin O) and immunohistochemistry for cartilage markers (collagen type II, Sox9, proteoglycan), proliferation-associated protein (Ki67) and markers of connective tissue (collagen type I and actin).

RESULTS:

The provision of a more natural microenvironment in vitro via co-culture of chondrocyte-based aggregates with osteoblast-like Saos-2 cells enhanced the differentiation potential of chondrogenic spheroids towards hyaline cartilage.

CONCLUSIONS:

The study showed the positive influence of Saos-2 cells on the differentiation potential of human chondrocytes in co-culture.

Keywords

Human chondrocytes Saos-2 HFF-1 co-culture spheroid scaffold-free

1 Introduction

Tissue engineering has become a major field of research in biotechnology, biomedicine, experimental pharmacology and basic research. First tissue engineered medicinal products have received the approval for use in hospitals for the regeneration of lost or destroyed tissue [1, 2]. Six out of ten biotechnologically processed tissue products are cell-based drugs to regenerate traumatic cartilage defects [1]. These efforts to heal cartilage damages are based on the fact that self-repair of this bone-coating tissue in the joints is very limited. However, cartilage defects are not limited to traumatic events. Also degenerative processes that result in osteoarthritis (OA) are a major public health problem in the world. OA affects 240 million people globally, about 10% of men and 18% of women over 60 years, leading to mobility deficits, chronic pain, reduced quality of life and contributing to mortality especially in people with age of 80 and older [3]. The disease is primarily characterized by articular cartilage degeneration, followed by subchondral bone thickening, osteophyte formation, synovial inflammation and joint degeneration [4]. Cartilage tissue, bone tissue and menisci show inflammatory degradation processes. During the inflammatory process, the bone cells (osteoblasts) secrete growth factors of the TGF-ß superfamily (TGF-ß and BMP) in order to positively influence the cartilage formation (chondrogenesis) of the affected tissue locations.

The close apposition of cartilage and bone in joints and the close interaction of chondrocytes and osteoblasts during cartilage and bone development in the process of endochondral ossification suggest that they may each modulate the other’s growth and differentiation [5]. Such an in vivo microenvironment of cells plays an important role to establish a milieu of biochemical, biomechanical and chemical signals connecting cells, extracellular matrix (ECM) and soluble factors [6, 7]. In vitro such a complex setup, even though ideal, is extremely difficult to realize. Co-culture technologies would be a method of choice to improve the cultivation success for mono-cultures and study cell-cell interactions between cell types in vitro [8]. This special technology enables also the delivery of biological, chemical and physical signals between different related cell types [9]. In contrast, a simple mono-culture operating with one cell type is based on the special conditions of this cell type for either cell proliferation or differentiation. To establish an appropriate microenvironment to optimize the culture conditions of cartilage cells and the formation of cartilage-like tissues in vitro could be the combination of chondrocytes and osteoblasts in a well-defined co-culture system. However, first analysis of differentiating parameters in chondrocyte microtissues in co-culture with osteoblasts are controversial depending on the co-culture system (direct versus indirect co-culture), the cells (mature versus immature chondrocytes versus stem cells, different species), and the culture type (monolayer versus microtissues) used [5 , 11].

This study investigates whether a more natural cartilage microenvironment could positively influence the chondrogenic tissue engineering process in vitro. Chondrocyte-only microtissues (mono-culture) showed an enhanced differentiation potential in vitro by co-cultivating chondrocytes isolated from the knee joint of adult donors with osteoblast-like osteosarcoma cells (Saos-2). The process of cell isolation and propagation in monolayer culture lead to the dedifferentiation of chondrocytes resulting in a kind of chondroprogenitors [12, 13]. Bearing in mind that the condensation of cells is the starting event during embryogenesis for the differentiation of mesenchymal stem cells into chondrocytes, three-dimensional aggregates preformed from monolayer-expanded chondrocytes are cultured in combination with Saos-2 cells or fibroblasts (HFF-1) in a transwell system [14].

2 Material and methods

2.1 Isolation of chondrocytes from human cartilage tissue

Human articular cartilage sample was obtained from the femoral condyle of a patient undergoing total knee surgery. An informed, written consent was obtained from the patient. There are no ethical concerns about the implementation of the project (ethics vote no. EK2017-8 of the ethics committee of the Brandenburg University of Technology Cottbus-Senftenberg). Isolation of chondrocytes was performed as previously described [15]. Briefly, chondrocytes were isolated by mechanical mincing of the tissue with a scalpel followed by enzymatic treatment (collagenase, 350 U/ml in DMEM:HAM’s F12 (1:1), Biowest, Nuaill^′e, France). The closed tube was placed on a shaker (Thermomixer comfort, Eppendorf, Hamburg, Germany) at 300 rpm interval mixing and incubated at 37°C for 20 h. The isolated chondrocytes were centrifuged at 300 xg for 5 min. The supernatant was removed and the cell pellet was resuspended in 10 ml of DMEM:HAM’s F12 (1:1) with 4 mM L-glutamine (Biowest) and 10% human serum (German Red Cross, Cottbus, Germany). The chondrocytes were cultured as monolayers in a humidified atmosphere at 37°C and 5% CO₂. Cells were detached for subculture using 0.05% trypsin/0.02% EDTA (Biowest), and plated at a defined ratio (1:3).

2.2 Generation of spheroids

Spheroids were generated using a scaffold-free culture system [15, 16]. Human chondrocytes (passage 2) were seeded in agarose coated 96-well plates at a cell density of 300,000 cells/well and cultured in two different differentiation media (DM), DMEM high glucose:HAM’s F12 (1:1) with 4 mM L-glutamine and 5% human serum (DM 1) and DM 1 plus ITS Liquid Media Supplement (1:100, Sigma-Aldrich GmbH, Steinheim, Germany) (DM 2). The morphology of the microtissues was documented using phase contrast microscopy (CKX 41 with a DP 71 camera, Olympus, Hamburg, Germany) and the size of the spheroids was measured using CellD-Imaging software for Life Science Microscopy (Soft Imaging Systems, Muenster, Germany).

2.3 Characterisation of co-culture cells in monolayer culture

Fibroblasts (HFF-1, ATCC SCRC-1041) as well as osteoblast-like osteosarcoma cells (Saos-2, ATCC HTB-85) were cultured under standard conditions in a humidified atmosphere at 37°C and 5% CO₂. The standard culture medium for HFF-1 cells is DMEM high glucose supplemented with 15% FBS (Biowest) and 4 mM L-glutamine, Saos-2 cells were cultured in McCoy’s 5A supplemented with 15% FBS. In co-cultures, both cell types (chondrocytes and HFF-1 or Saos-2, respectively) must be cultured in the same medium. To analyze the influence of the chondrocyte differentiation media DM 1 and DM 2 on the proliferation of Saos-2 and HFF-1 cells growth curve experiments were performed. Briefly, Saos-2 cells and HFF-1 cells were seeded in 6-well plates at a cell density of 4×10³ cells per cm² and cultured for 3 weeks. The cells in the wells were counted each day and the cell numbers per cm² were plotted logarithmically over time.

2.4 Co-culture of microtissues with Saos-2 cells and HFF-1 cells

For co-culture experiments Saos-2 cells and HFF-1 cells were seeded on tissue culture inserts (suitable for 24-well plates, pore size 3μm, Greiner Bio-One, Kremsmünster, Austria) 24 h prior to the co-culture start to allow full adherence. 10-day old spheroids were transferred into agarose-covered 24 well plates. The tissue culture inserts were placed above the spheroids, and incubated for 27 days with medium change every 2 to 3 days. During the cultivation period, the morphology and size (diameter) of the spheroids were documented using the OLYMPUS CKX41 phase contrast microscope (10 individual spheroids for each time point and each co-culture setup). At the end of cultivation the macroscopic appearance was documented using the OLYMPUS SZX10 reflected-light microscope and a DP 71 camera.

2.5 Histological and immunohistochemical analysis

Spheroids were analysed after 10 days of mono-culture in DM 1 and DM 2 and after additional 27 days of co-culture with HFF-1 and Saos-2 cells, respectively. The in vitro microtissues were harvested, embedded in Neg-50 frozen section medium (Richard Alan scientific, Kalmazoo, USA) and sectioned using a cryomicrotom (Microm GmbH, Walldorf, Germany). Cryosections on glass slides were fixed in a two-step process. A formalin fixation (4% at 4°C for 10 min, AppliChem, Darmstadt, Germany) was followed by incubation in a mixture of methanol/acetone (1:1 at 20°C for 10 min, Roth, Karlsruhe, Germany). Histological staining was performed with Safranin O-Fast Green (SO) (AppliChem) and Alcian-Blue 8 GS (Serva Electrophoresis GmbH, Heidelberg) to visualize proteoglycans and glycosaminoglycans [15, 17]. Tissue nonspecific alkaline phosphatase was stained using the Alkaline Phosphatase Kit from Sigma-Aldrich. Analyses of histological preparations were done using a BX41 microscope (Olympus, Hamburg, Germany) equipped with a Color View I camera (Olympus) and CellD-Imaging software.

Immunohistochemical analyses were carried out to detect human collagen type I, collagen type II, cartilage proteoglycan, Sox-9, and Ki-67 in fixed cryosections of spheroids and native human articular cartilage from the original condyle (positive/negative control). Sections were rinsed with phosphate-buffered saline (PBS) and incubated for 20 min at room temperature (RT) with normal goat serum (Dianova, Hamburg, Germany) diluted 1:50 in PBS/0.1% BSA (Roth). Primary antibodies were diluted in PBS/0.1% bovine serum albumin (BSA) as follows: anti-collagen type I (1:800) and anti-collagen type II (1:600) (MP Biomedicals, Ohio, USA), anti-cartilage proteoglycan (1:100) and anti-Sox-9 (1:500) (Millipore, Temecula, USA), anti-Ki67 (1:50, DakoCytomation, Glostrup, Denmark). The cryosections were incubated with primary antibodies in a humidified chamber overnight at 4°C. After washing three times with PBS, the slides were incubated for 1 h at RT with Cy3-conjugated goat anti-mouse (collagen Type I/II, cartilage proteoglycan, Ki-67) and goat anti-rabbit (Sox-9) antibody (Dianova) diluted 1:600 in PBS/0.1% BSA including DAPI (1 mg/ml; Fluka, Seelze, Germany) to stain cell nuclei. The preparations were mounted in fluorescent mounting medium (DakoCytomation) and analyzed by fluorescence microscopy (OLYMPUS IX81) equipped with a Retiga 6000 (QImaging, Surrey, BC, Canada) camera and CellSens 1.14 imaging software (Olympus). In order to test for unspecific binding of the secondary antibody, staining without primary antibody was included in all experiments. Cryosections of the original condyle were used as positive control for collagen type II, proteoglycan, actin and Sox-9 and as negative control for Ki-67 and collagen type I.

The cytoskeleton element actin was visualized using Alexa Fluor^® 555-conjugated Phalloidin (1:150) (Millipore) after fixation with 4% formaldehyde (RT for 10 min) and washing with PBS.

2.6 Statistical analysis

The results are presented as mean±SEM (standard error of mean) of cells from one donor in triplicate measurements. Statistical significance was analyzed by two-way ANOVA with Bonferroni’s post-test and is indicated by *** (p < 0.001).

3 Results

3.1 Optimization of cell culture conditions for the spheroid aggregation process

The differentiation potential of human chondrocytes isolated from a femoral condyle and proliferated in monolayer culture were tested in relation to two different media. Differentiation medium one (DMEM/Ham’s F12, 5% human serum), a known standard medium for chondrogenic differentiation, was compared to differentiation medium two (DMEM/Ham’s F12, 5% human serum, ITS), which contained additionally to DM 1 the supplements insulin, transferrin and sodium selenite. Figure 1 shows the size of the spheroids within the first 10 days of the aggregation process in mono-culture. Cells cultured in DM 1 were significantly (p < 0.001) reduced in size. Both media supported the formation of round sphere-like aggregates (phase contrast images).

Fig.1

Aggregation process of spheroids in mono-culture. Chondrocytes (3×10⁵ cells/well) were seeded in agarose-covered 96-well plates and cultured for 10 days. The average diameter (μm) was calculated from 10 individual spheroids (±SEM). Phase contrast pictures were taken at day 10. Scale bar: 1000μm.

After 10 days of mono-culture the spheroids were analyzed histochemically (Alcian Blue, Safranin-O) and immunohistochemically (IH: collagen type I/II, proteoglycan, Sox-9, Ki-67) for standard chondrogenic markers.

ITS treated aggregates showed an increased assembly of proteoglycans (Fig. 2: light Alcian Blue staining, Fig. 3: anti-human cartilage proteoglycan antibody) and a higher expression of the early chondrogenic transcription factor Sox-9 (Fig. 3). Collagen type II, the main collagen type in cartilage, is positive in the original condyle but it is not visible in these young aggregates via IH. Collagen type I is not typical for hyaline cartilage and at the best found at the outer rim of the joint surface of the cartilage tissue. The chondrocytes in these young aggregates treated with ITS exhibit in some parts collagen type I positivity.

Fig.2

Histochemical analyses of spheroids after 10 days in mono-culture. Comparison of DM 1 and DM 2. Positive controls are cartilage tissue sections of the original condyle. (A) Safranin O-staining of cryosections (red: proteoglycans; dark blue: cell nuclei). (B) Alcian Blue staining of cryosections (blue: glycosaminoglycans; red: nuclei). Scale bar: 100μm.

Fig.3

Immunohistochemical analyses of cartilage-like mono-culture spheroids after 10 days. Comparison of two differentiation media (DM 1, DM 2). Controls are cartilage tissue sections of the original condyle. Indirect immunofluorescence for collagen type II (A), proteoglycan (B), Sox-9 (C), Ki-67 (D) and collagen type I (E) (red: Cy-3 conjugated antibody; blue: cell nuclei). Actin (F) was visualized using phalloidin (red: Alexa Fluor^® 555-conjugated phalloidin; blue: cell nuclei). Scale bar: 50μm.

Ki-67, a cellular marker for proliferation, is negative. This is expected, because chondrocytes undergoing differentiation stop to proliferate. The actin staining is positive in all three samples. Comparing the results shown in Figs. 2 and 3 demonstrate that the addition of ITS to the medium enhances the differentiation capacity of the chondrocytes in young mono-culture spheroids.

3.2 Behavior of co-culture cells in different media

To evaluate the influence of medium with/without ITS on the proliferation of osteoblast-like Saos-2 cells and fibroblast-like HFF-1 cells we performed growth curve analysis. Figure 4 shows the growth curves of Saos-2 cells and HFF-1 cells in DM 1 and DM 2 in comparison with the standard medium for each individual cell line. Both cell types showed no significant change in proliferation (and also morphology, data not shown) in the different culture media. Therefore, the following co-culture experiments were performed with DMEM/Hams’ F12, 5% human serum and addition of ITS (DM 2).

Fig.4

Growth curves of osteoblast-like Saos-2 cells and fibroblasts (HFF-1 cells). Cells (seeding density 4×10³ cells/cm²) were cultured in DM 1, DM 2, and the standard medium for each individual cell line for 22 days.

3.3 Chondrogenic microtissues in co-culture show an altered differentiation profile

Co-cultures of chondrocyte-based spheroids and osteogenic cells or fibroblasts as partners vary greatly in size after 37 days of total cultivation time. Figure 5 summarized the size development of the three conditions. Spheroids co-cultured with Saos-2 cells had the largest diameter (880μm) followed by chondrocytic spheroids in mono-culture (740μm) and the smallest microtissues were formed of spheroids co-cultured with HFF-1 cells (631μm). The macroscopic analysis of the spheroids showed sphere-like solid aggregates in differing sizes with a broader transparent outer rim present in the co-culture with Saos-2 cells.

Fig.5

Size development of co-culture spheroids. 10-day old spheroids in DM 2 were transferred in agarose-covered 24-well plates and cultured in the presence of Saos-2 cells and HFF-1 cells on tissue culture inserts. The average diameter (μm) was calculated from 10 individual spheroids (±SEM). Stereomicroscopic images were taken at day 37. Scale bar: 500μm.

Spheroids co-cultured with Saos-2 cells showed an increased assembly of proteoglycans (Fig. 6: Alcian Blue staining and Safranin O staining, Fig. 7: IH using anti-human cartilage proteoglycan antibody), an elevated production of collagen type II (Fig. 7), and a higher expression of the early chondrogenic marker Sox-9 in comparison to spheroids in mono-culture. Co-culture of spheroids with HFF-1 cells even resulted in a lower expression of theses chondrogenic markers compared to spheroids in mono-culture. Collagen type I is negative in all three culture condition.

Fig.6

Histological analyses of cartilage-like spheroids in co-culture after 37 days. Comparison of spheroids in mono-culture with spheroids in co-culture with HFF-1 cells and with Saos-2 cells. (A) Safranin O-staining of cryosections (red: proteoglycans; dark blue: cell nuclei). (B) Alcian Blue staining of cryosections (blue: glycosaminoglycans; red: cell nuclei). (C) Staining of tissue nonspecific alkaline phosphatase (red: TNAP). Scale bar: 100μm.

Fig.7

Immunohistochemical analyses of cartilage-like spheroids in co-culture after 37 days. Indirect immunofluorescence for collagen type II (A), proteoglycan (B), Sox-9 (C), Ki-67 (D) and collagen type I (E) with respect to different co-culture conditions (red: Cy-3 conjugated antibody; blue: cell nuclei). Actin (F) was visualized using phalloidin (red: Alexa Fluor^® 555-conjugated phalloidin; blue: cell nuclei). White lines indicate the border between chondrocytes (concave side) and membrane-permeable Saos-2 cells attached to the spheroid (convex side). Scale bar: 50μm.

The histochemistry (Fig. 6) of the tissue non-specific alkaline phosphatase (TNAP), a standard marker for osteoblast-like Saos-2 cells, and the immunhistochemistry of the proliferation marker Ki-67 showed that Saos-2 cells were able to penetrate through the tissue culture inserts. We found a layer of Saos-2 cells as an outer rim of the co-cultured spheroids. This is visualized in Fig. 7 with the white lines dividing chondrocytes (concave side) and Saos-2 cells (convex side). The actin staining is positive in all three samples with an intense and structured staining of the Saos-2 cells. Summarizing our data we can conclude that co-culture with osteoblast-like Saos-2 cells enhances the differentiation potential of chondrogenic spheroids towards hyaline cartilage.

4 Discussion

The interaction of cartilage and bone is of decisive importance for the movement processes of the body. At the cellular level, chondrocytes of the cartilage meet the osteoblasts of the bone. These natural cell-cell interactions are part of the development of the articular cartilage and its environment in vivo. In vitro cell culture systems should therefore consider direct co-cultures of chondrocytes and osteoblast or indirect co-culture with the cell types separated via a membrane to allow a two-way medium exchange [14]. The combination of sphere-like aggregates of chondrocytes with osteoblast-like or fibroblast-like monolayer cultures in a transwell system allows the study of medium dependent influences between those cell types in an in vivo-like tissue model. Aggregates used for co-cultures should be optimized with respect to the components used [18]. To avoid any influence of a biomaterial on the cell-cell crosstalk in co-culture [14 , 20] a scaffold-free spheroid system was selected [15, 16]. An optimal cell number per spheroid was determined as previously described [21] and a suitable medium composition was identified. Best cell aggregation and start of differentiation could be observed in medium containing ITS (Figs. 1, 2, 3). Based on these results, medium with ITS (DM 2) was used for the following co-culture experiments.

Saos-2 cells, a characterized human cell line with osteoblastic properties, was selected for the co-culture experiments. Malignant cells often express differentiated features of the tissue of origin along with cellular immortality. Established cell lines might combine these properties and make it possible to study phenotype-related cellular functions [22]. Furthermore, the selected co-culture cells should not change their normal behavior in the medium selected for the combined culture [23]. Saos-2 and HFF-1 cells presented an identical morphological appearance in monolayer culture and hardly any variations in growth behavior (Fig. 4).

Combining these pre-characterized partners in a transwell co-culture system revealed a positive influence of osteogenic cells on the differentiation of chondrocytic spheroids (Figs. 5, 6, 7). Microtissues after 4 weeks in co-culture with Saos-2 cells were bigger in size and showed an increased expression of cartilage markers like specific ECM molecules (proteoglycans, collagen type II) and the chondrogenesis regulating transcription factor Sox-9 [24]. The connective tissue and bone specific collagen type I was not detected via immunohistochemistry, a further hint to a shift to a chondrogenic phenotype of the dedifferentiated chondrocytes from monolayer culture expressing collagen type I (data not shown) [25]. This phenotypic shift was supported by a missing Ki-67 expression, a proliferation associated protein also expressed in the monolayer expanded chondrocytes (data not shown).

The aim of the experimental set up was to establish an indirect co-culture separating chondrogenic microtissues and the corresponding osteoblastic co-culture cells via a membrane to study a possible effect of paracrine signalling. However, during the cultivation period the Saos-2 cells were able to pass the tissue culture inserts with pore size of 3μm resulting in a direct co-culture. The osteoblastic cells formed both cellular monolayers up to a multi-layered tissue coating. Saos-2 cell“coatings” around the chondrogenic microtissue were visualized via the osteoblast specific TNAP staining (tissue nonspecific alkaline phosphatase, Fig. 6) [26], as well as by excluding the expression of cartilage-specific markers (Fig. 7). In addition, Saos-2 cells are positive for Ki-67, as they proliferate also in cell groups or microtissues (Fig. 6) based on their tumor origin. They also express high amounts of the cytoskeleton element actin, which is well detectable in the large Saos-2 cells by phalloidin.

Chondrogenic differentiation could be induced or augmented by direct cell-cell interaction (e.g. N-cadherin, N-CAM, gap junctions), signalling molecules like growth factors (TGF-β family, BMPs, IGF-I), or hormones (e.g. PTHrP, parathyroid hormone-related peptide) [27]. Saos-2 cells express some of these soluble factors which include bone morphogenetic proteins (particularly BMPs-2, 3, 4, and 7) and also transforming growth factor-beta (TGF-β) [28] which are also known to be secreted by the subchondral bone in joints [29]. The combination of BMPs and TGF-β increases cartilage-specific markers (collagen type II, proteoglykans), while the expression of collagen type I seems to be minimal [30, 31] A whole range of factors may be exchanged during the co-culture of chondrocyte-based microtissues and osteoblast-like cells and might therefore be involved in the augmentation of chondrogenesis. However, this experimental approach lacks the ability to explore the complex interactions that take place between the multiple paracrine and autocrine signals between cartilage and bone cells.

5 Conclusion

The study showed a positive influence of osteoblast-like Saos-2 cells on the differentiation potential of human chondrocytes in 3D culture. Additional experiments are planned to further evaluate the data and to investigate the donor dependent chondrogenic potency in correlation to the co-culture effects of osteoblasts and chondrocytes. Additionally, this co-culture system may be suitable to study the development and applicability of transplants for a cell-based therapy for osteochondral defects.

Footnotes

Acknowledgments

The work was supported by grants of the “Gesundheitscampus Brandenburg” and the “Ministerium für Wissenschaft, Forschung und Kultur” (Land Brandenburg, Germany).

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