Enhanced Superficial Zone Chondrocyte Expansion and Redifferentiation by Culture on Chondrocyte-Derived Decellularized Matrices

Abstract

Background

Cell-based therapies to regenerate native-like cartilage are limited by the inability to re-express zone-specific molecules. While monolayer-expanded (passaged) chondrocytes are a clinically approved cell source, the resulting tissues have reduced Proteoglycan-4 (PRG4) expression. This may be due to poor attachment, slow proliferation, and dedifferentiation of superficial zone chondrocytes (SZC) on polystyrene. Optimizing expansion conditions is therefore critical. Chondrocyte-derived decellularized extracellular matrix (CD-ECM) has been shown to enhance proliferation and reduce dedifferentiation of full-thickness chondrocytes, but its effect on SZC remains unknown. We tested the hypothesis that culturing SZC on CD-ECM would improve attachment, proliferation, and reduced dedifferentiation, enabling formation of PRG4-expressing tissue.

Methods

Primary bovine SZC were seeded on polystyrene or CD-ECM. Attachment, expansion rate, and gene expression were evaluated during passaging. Cells from each condition were assessed for their capacity to form PRG4-expressing bioengineered tissue.

Results

Primary bovine SZC had increased attachment and reached confluency faster on CD-ECM. SZC on CD-ECM were smaller, with fewer actin stress fibers, and exhibited reduced expression of dedifferentiation markers. Furthermore, SZC expanded on CD-ECM were stimulated to form tissues rich in Collagen II and Aggrecan with higher Proteoglycan-4 expression.

Conclusions

The use of CD-ECM for passaging SZC may aid in achieving an adequate number of SZC for bioengineering purposes.

Keywords

superficial zone chondrocytes dedifferentiation proteoglycan-4 extracellular matrix decellularized matrix redifferentiation bioengineered cartilage

Introduction

Articular cartilage matrix is primarily made up of proteoglycan (mostly Aggrecan; ACAN) and collagen (mostly Collagen II; COL2), with depth dependent zonal organization containing chondrocyte populations that contributes to biological and mechanical properties of articular cartilage. A critical function of articular cartilage is to provide a lubricated surface for joint movement and to facilitate mechanical load transmission onto underlying bone.^1,2 The superficial zone chondrocytes (SZC) residing in the superficial zone of cartilage and produce a critical lubricating molecule, Proteoglycan-4 (PRG4). PRG4 is an essential component of a frictionless fluid-like layer at the tissue surface, which enables cartilage to resist biomechanical forces generated during articulation, such as shear and compression.^3
-6 When articular cartilage is damaged, degeneration is initiated leading to osteoarthritis progression. The progression of osteoarthritis leads to the breakdown of the surface of articular cartilage, causing loss of frictionless motion and further cartilage damage.^7,8 Bioengineered cartilage serves as a promising methodology to repair cartilage defects and prevent Osteoarthritis progression. For successful repair of cartilage, it is imperative that the generated bioengineered tissue with adequate PRG4 expression as the expression of PRG4 in repair tissue enhances the wear-resistance properties of tissue leading to improved repair outcomes.⁹

Cell-based therapies aim to repair small cartilage defects by implanting cells directly into the damaged area to slow osteoarthritis progression and preventing further cartilage degradation.^10,11 Monolayer expanded (passaged) full-thickness chondrocytes are an FDA approved cell-source for cartilage repair therapies and have been previously used to generate bioengineered cartilage.^10
-12 However, the use of passaged chondrocytes for cartilage repair has limitations. 2D monolayer cell expansion on polystyrene (PS) results in chondrocyte dedifferentiation. In dedifferentiation, chondrocyte morphology is altered from round to a spread and flattened appearance due to reorganization of the actin cytoskeleton.^13
-16 This reorganization drives fibrocartilage production in bioengineered cartilage by increasing the expression of fibroblast matrix molecules Type I Collagen (COL1) and Tenascin C (TNC) and contractile molecules such as Alpha Smooth Muscle Actin (αSMA), and decreased expression of chondrogenic expression for COL2 and ACAN.^13,14,17
-19

Passaged chondrocytes are capable of redifferentiation in 3-dimensional (3D) culture, allowing for the re-expression of their chondrogenic phenotype, enabling the production of native-like bioengineered cartilage.²⁰-²³ Previously, we have shown that passaged full-thickness chondrocytes are capable of producing bioengineered tissue rich in COL2 and ACAN via culture in a 3D scaffold-free culture, supplemented with chondrogenic media.²³ While current methodologies allow for the generation of bulk cartilage matrix by redifferentiation of passaged chondrocytes, a challenge that remains is the generation of a superficial zone by passaged cells. As compared with native cartilage, bioengineered tissue derived from redifferentiated passaged full thickness cells produce low amounts of PRG4.^22
-24 The limited ability to produce PRG4 could be due monolayer expansion, which decreases the expression of PRG4.^22,23 Furthermore, there are additional challenges in obtaining adequate numbers of SZC for bioengineering purposes. It has been previously shown that while both passaged SZC and deep zone chondrocytes can produce matrix containing ACAN and COL2, the accumulation of ACAN and COL2 is much greater by redifferentiated deep zone chondrocytes.²¹ Interestingly, PRG4 production by passaged cells requires cells that originate from the superficial zone; redifferentiated deep zone cells do not produce PRG4.^21,22 However, the superficial zone of articular cartilage is the first to wear during OA progression. Thus, full thickness chondrocytes harvested from a donor-site may have a limited number of SZC. Second, previous studies have found that SZC have poor primary attachment onto PS. Therefore, many cells that attach to PS may be representative of deeper zone chondrocytes. Third, SZC proliferate slower on PS compared with chondrocytes from other zones.^22,25 Overall, these findings suggest that traditional methods of passaging full-thickness chondrocyte-based therapies results a low proportion of SZC left in culture, leading to limited PRG4 expression in the bioengineered tissue. Therefore, it is imperative to optimize expansion methods to obtain an adequate number of SZC for bioengineering purposes.

In articular cartilage, the extracellular matrix (ECM) provides a combination of biophysical, biomechanical, and biochemical cues that regulates chondrocyte proliferation, adhesion, migration, and differentiation.^26
-31 The use of decellularized matrices that provide biological ECM cues serve as a promising substrate for in vitro expansion of cells. Both synovium-derived stem cells and mesenchymal stem cells derived decellularized matrices have been shown to support the differentiation of mesenchymal stem cells into chondrocytes.^30,31 Previously, it has been shown that passaged human chondrocyte derived extracellular matrices (CD-ECM) support the cell number expansion of full-thickness primary human chondrocytes. The use of CD-ECM was found to increase proliferation rate, reduce chondrocyte dedifferentiation, and promote tissue formation with lower expression of COL1 in the matrix.²⁸ It remains to be determined if CD-ECM could also be used to enhance SZC attachment, proliferation, and redifferentiation.

This study investigates whether CD-ECM can enhance the therapeutic potential of SZC for cartilage repair. We hypothesize that CD-ECM promotes greater attachment and expansion of SZC while preserving their phenotypic characteristics, leading to improved capacity for forming superficial zone-like cartilage tissue rich in PRG4 expression. The overarching objective is to addresses a critical barrier in cartilage tissue engineering, in obtaining an adequate subpopulation of SZC. The outcomes of this study may support the development of more effective cell-based therapies for joint surface regeneration, particularly in re-establishing the lubricating superficial zone, which is essential for long-term cartilage function and integrity.

Methods

Generation of CD-ECM Substrates

CD-ECM (CELLvo™ ChondroMatrix) substrates were generated using previously described methods, with slight modifications.²⁸ Healthy human articular cartilage sample from a young and otherwise healthy cadaver was obtained (GenCure Tissue Services, BioBridge Global, San Antonio, TX, USA). As the tissue is cadaveric tissue for Research Use Only, it is therefore not governed by the Institutional Review Board in the United States. Institutional Review Board oversight is not required. Human articular chondrocytes were harvested as previously described.²⁸ These cells were later immortalized by Alstem (Richmond, CA, USA). 1 x 10⁵ cells were infected with lentivirus encoding SV40 with puromycin resistance gene. The cells were selected by puromycin at the concentration of 2 μg/ml for 3 days and continued to passage for about 3 passages. The cells were expanded and frozen.

To produce matrix, the immortalized cells were thawed and seeded onto fibronectin-coated PS and cultured to confluence in DMEM supplemented with 10% fetal bovine serum (FBS). At confluence, the medium was supplemented with 50 µM ascorbic acid to induce matrix protein secretion. After 24 to 72 hours, the resulting ECM was washed once with PBS to remove media and then incubated for 7 min at room temperature with PBS containing 0.5% (v/v) Triton X-100 with 20 mM NH₄OH to decellularize the ECM. The decellularized ECM was washed thoroughly with PBS and deionized water to remove the detergent before being allowed to air dry, as previously described.²⁸ The dried plates, coated with intact decellularized ECM, were stored at 4 °C until use. Before use, dried plates were rehydrated with PBS at 37 °C for 1 h.

Decellularization was confirmed in 2 different ways. First, cell seeded, and non-seeded ECM-Substrates were stained with Hoechst 33342 (Nuclei: 1/500; Biotium, Fremont, CA, USA), rhodamine-phalloidin (F-Actin; 1:50; Biotium), and Wheat Germ Agglutinin CF640R Conjugate (CD-ECM: Biotium) to visualize any remanent cells or DNA (Suppl. Fig. S1A). Second, DNA content of CD-ECM was analyzed via papain digestion solution, 40 mg/ml papain (Roche) in 20 mM ammonium acetate (Fisher Scientific, Hampton, NH, USA), 1 mM EDTA (Fisher Scientific), and 2 mM dithiothreitol at pH 6.2, and digested for 4 hours at 65 °C. DNA content from papain was quantified using the Hoechst 33258 (ABCAM, Cambridge, United Kingdom) dye-binding assay. A standard curve was generated using calf thymus DNA (Sigma-Aldrich) diluted in PBS−/−. Absorbance was measured using fluorescent spectrophotometry (Glomax Multi+ Detection System; Promega, Madison, WI, USA; excitation, λ = 355 nm; emission, λ = 460 nm) (Suppl. Fig. S1B).

Bovine SZC Isolation and Expansion

SZ cartilage was manually microdissected from Bovine metacarpal-phalangeal joints as previously described.^22,32 Chondrocytes were isolated from the SZ cartilage by serial enzymatic digestion in 0.5% protease (Sigma-Aldrich) for 45 minutes at 37 °C, followed by 0.1% collagenase (Roche, Mannheim, Germany) for 14 to 17 hours at 37 °C. Once isolated, the harvested SZC and the remaining underlying chondrocytes were evaluated to ensure selective isolation of SZCs (Suppl. Fig. S2). For monolayer expansion of SZC, cells were seeded at 1500 cells/cm² on either polystyrene (Corning, Edison, New Jersey, USA) or CD-ECM. Cells were serially passaged in expansion media (Hams F-12 media [Corning] supplemented with 10% FBS (GenClone, Genesee Scientific, San Diego, CA, USA) and 1% antibiotic-antimycotic (Corning)). Chondrocytes were cultured until approximately 80% to 90% confluent and then detached with 0.25% trypsin-EDTA (GenClone) and designated passaged 1 (P1) cells. P1 SZC from PS or CD-ECM were reseeded in monolayer (2 x 10³ cells/cm²) on their respective surface. Cells were cultured until approximately 80% to 90% confluent and then detached with 0.25% trypsin-EDTA. The detached cells were designated as P2 cells.

Cell Attachment Assays

The attachment of SZC onto CD-ECM was compared with PS via 2 ways. First, image quantification of SZC coverage on respective substrates. Second, by quantifying DNA as a proxy of cell number on respective substrates.

To quantify SZC coverage, P0 SZC were seeded at 5.0 x 10⁴ cells/cm² in monolayer on either PS or CD-ECM, in expansion media. After 24 hours, the media was removed, and cultures were washed with PBS−/−, Without calcium and magnesium (−/−); Quality Biological, Gaithersburg, MD, USA, to remove nonadherent cells. Light microscopy images were acquired using a Zeiss Primovert tissue culture microscope (Zeiss, Jena, Germany) with an attached camera (Swiftcam Technologies; Hong Kong, China). Images were imported into FIJI (ImageJ; National Institutes of Health), and individual cells were manually traced using the freehand selection tool. The total traced cell area was calculated and divided by the total image area to determine the percent area covered by cells. At least 3 wells were analyzed per experimental set.

To quantify DNA of cells on substrates, P0 SZC were seeded at 5.0 x 10⁴ cells/cm² in monolayer in expansion media on either PS or CD-ECM. After 24 hours, the media were removed, and cultures were washed with PBS−/− to remove nonadherent cells. The attached cells were harvested via papain digestion solution (40 mg/ml papain in 20 mM ammonium acetate, 1 mM EDTA, and 2 mM dithiothreitol at pH 6.2) and digested for 4 hours at 65 °C. The DNA content of attached cells on each culture surface was compared with the DNA content of initial cell aliquots. DNA content from papain digested cell pellets was quantified using the Hoechst 33258 dye-binding assay. A standard curve was generated using calf thymus DNA diluted in PBS−/−. Absorbance was measured using fluorescent spectrophotometry (Glomax Multi+ Detection System; excitation, λ = 355 nm; emission, λ = 460 nm).

Confluency

Light microscopy images were acquired using a Zeiss Primovert tissue culture microscope under consistent lighting and magnification settings. Images were captured daily to monitor cell growth and confluency. To quantify confluency, images were analyzed using FIJI software. Individual cells were manually traced using the freehand selection tool to accurately identify the cell-covered area in each image. The total cell area was then divided by the total image area to calculate the percentage of confluency. This analysis was performed on SZC cultures each day during passaging from P0 to P1 and from P1 to P2, allowing comparison of cell expansion over time on different substrates.

RNA Extraction and RT-PCR

Total RNA was extracted from cells and tissues using TRIzol reagent (Sigma-Aldrich), following the manufacturer’s instructions. RNA purity and concentration were assessed using a NanoDrop One spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA), based on A260/A280 and A260/A230 ratios. Samples were reverse transcribed to cDNA using UltraScript 2.0 cDNA Synthesis Kit (PCR Biosystems; Wayne, PA, USA) according to the manufacturer’s directions. Quantitative real-time PCR (qPCR) was performed using 20 ng of cDNA per reaction, gene-specific primers (10 μM working concentration; sequences, GenBank accession numbers, and temperature provided in Suppl. Table S1), and qPCRBIO SyGreen Blue Mix (PCR Biosystems). All primer pairs were designed using NCBI Primer-BLAST. Reactions were run in technical duplicate on a Cielo 3 Real-Time PCR System (Azure Biosystems, Houston, TX, USA). No-template controls (NTCs) and –RT controls were included in each run to ensure specificity and lack of contamination. The Pfaffl method was used to calculate mean relative quantification values (NRQ) using 18s rRNA as the endogenous housekeeping gene.³³ DNA contamination was assessed by examining the A260/A280 ratio and further confirmed by running a no-reverse transcriptase control (–RT) in the qPCR analysis.

Area and Circularity

P0 and P2 SZC were seeded at 5.0 x 10⁴ cells/cm² in monolayer on PS and CD-ECM in expansion media. 24 hours after seeding, light microscopy images were taken on a Zeiss Primovert tissue culture microscope with an attached camera (Swiftcam). FIJI was used to manually trace the boundaries of adhered cells. Area and circularity were measured as previously described.^15,34 At least 3 image fields were analyzed, totaling a minimum of 45 cells per condition.

Confocal Imaging of Actin Organization and Polymerization

P2 SZC passaged on PS or CD-ECM were seeded at 1.0 x 10⁴ cells/cm² on non-coated or CD-ECM-coated Thermanox Plastic coverslips (Thermofisher). After 24 hours, cells were fixed by incubating cells in 4% paraformaldehyde (Electron Microscopy Sciences, Hatfield, PA, USA) at room temperature for 15 minutes, and were then washed 3 times with PBS−/−. Cells were then permeabilized using permeabilization/blocking solution (3% Goat serum, 3% BSA, and 0.3% Triton) for 30 min. To visualize globular (G-) and filamentous (F-) actin, P2 SZC on PS and CD-ECM coverslips were stained with vitamin D binding protein conjugated to Alexa 488 (G-actin; VitDBP-488; 1:100; RayBiotech, Peachtree Corners, GA, USA) and rhodamine-phalloidin (F-Actin; 1:50; Biotium). Cells were counterstained with Hoechst 33342 (1/500; Biotium). Coverslips were then mounted using Drop-n-Stain mounting medium (Biotium). Confocal microscopy imaging was performed using a Zeiss LSM880 laser scanning confocal fluorescence microscope (Zeiss), Z-stack images were acquired using a 20x objective (NA: 0.4 oil-objective; 0.7-μm slices) or 63x objective (NA: 1.3 oil-objective; 0.3-μm slices).

The ratio of G/F-actin fluorescent intensity of P2 SZC on PS and CD-ECM was analyzed through FIJI as previously described.^34,35 Cell boundaries were traced using phalloidin staining of F-actin at the cell boundaries. Mean fluorescence intensities were quantified in each channel, and G/F-actin ratios were determined by dividing the mean fluorescent intensity of G-actin (Green; VitDBP-488) by the mean fluorescent intensity of F-actin (Red; rhodamine-phalloidin). The calculated G/F-actin fluorescence intensities for 15 cells per coverslip were averaged per coverslip. Values were normalized to the average values of the control group within each set. Each experiment has 3 coverslips per condition.

G/F-Actin Quantification via Triton Solubility

To determine actin polymerization status of P2 SZC, we performed Triton solubility assay.^34,36 Soluble portions (containing predominantly G-actin) were isolated by placing 0.1%Triton in cytoskeletal buffer (100 mM NaCl, 3 mM MgCl2, 300 mM Sucrose, 1 mM EGTA, 10 mM PIPES) onto cells for 2 minutes. This solution was removed from the wells and placed into microcentrifuge tubes containing RIPA (10x; EMD Millipore, Burlington, MA, USA). The remaining insoluble portions (containing predominantly F-actin) were isolated by placing the cells in 0.1% Triton in cytoskeletal buffer containing 1X RIPA. The remaining cell contents were scraped from dishes and placed in microcentrifuge tubes. The samples were then incubated on ice for 30 minutes. Samples were sonicated and equal volumes of the Triton-soluble and insoluble fractions were prepared for WES capillary electrophoresis. Protein was separated using a 12–230 kDa separation module (Protein Simple, San Jose, CA, USA) and actin was probed using a rabbit anti–pan-actin (1:100; Cell Signaling, Danvers, MA, USA) antibody. The proportion of G-actin, the Triton-soluble portion, was quantified by dividing the amount of Triton-soluble actin by total actin, giving a percentage of G-actin.

3D Redifferentiation Culture

P2 SZC passaged on PS and CD-ECM were seeded at high density in 3D scaffold-free culture within agarose molds in expansion media, as previously described.^22,23 To form the mold, molten agarose was poured into the wells of a 12-well culture plate. Once gelled, an 8-mm biopsy punch was used to punch out a mold. 2 x 10⁶ P2 SZC were then seeded within each agarose mold. After 48 hours, expansion media were replaced with redifferentiation media defined as Dulbecco’s modified Eagle medium (GenClone) supplemented with ITS 1 Premix (6.25 mg/ml insulin, 6.25 mg/ ml transferrin, 6.25 mg/ml selenium, 1.25 mg/ml bovine serum albumin, and 5.35 ng/ml linoleic acid) (Corning), 0.1 mM dexamethasone (Sigma-Aldrich), 40 mg/ml proline (Sigma-Aldrich), 110 mg/ml pyruvate, and 100 mg/ml ascorbic acid (Sigma-Aldrich). After day 5 of culture, cells were maintained in redifferentiation media supplemented with 10 ng/ml transforming growth factor beta 3 (TGFβ; R&D Systems, Minneapolis, MN, USA). Media was changed every 2 days until tissues were harvested at days 10 and 21.

Histology

Tissues harvested on day 21 were fixed in 10% buffered formalin (StatLab, McKinney, TX, USA; pH 7.4) overnight at 4 °C. Tissues were then embedded in paraffin and sectioned. Five-micron sections were stained with hematoxylin and eosin, toluidine blue, or picrosirius red and mounted. Sections were imaged using a Zeiss Axio Observer Inverted microscope (Zeiss).

Tissue Immunostaining and Quantification

Tissues were fixed in 4% paraformaldehyde overnight at 4 °C, washed with PBS−/−, and stored in 30% sucrose (Sigma-Aldrich) overnight at 4 °C. The tissues were then snap-frozen in OCT compound (Sakura Finetek, Torrance, CA, USA) and cryosectioned in 8-micron sections. Antigen retrieval was performed on sections by digestion with a solution of 0.4% pepsin (Sigma-Aldrich) in 1x Tris-buffered saline (pH 2). Sections are then blocked with 20% goat serum, and then incubated overnight at 4 °C with primary antibodies listed in Supplementary Table S2. After 24 hours, tissues were incubated with secondary antibodies listed in Supplementary Table S2 for 1 hour at room temperature and counterstained with Hoechst 33342 (1/500; Biotium). Tissues were washed 3 times with PBS−/− between incubations. Slides were then mounted with Drop-n-Stain mounting medium (Biotium) and glass coverslips. Confocal microscopy imaging was performed using a Zeiss LSM880 laser scanning confocal fluorescence microscope (Zeiss) with a 20x objective. Z-stack images were acquired using 0.7-μm slices.

To quantify total fluorescent intensity, images were analyzed on FIJI by tracing the edges of the tissues and measuring the fluorescent intensity of the channel corresponding to the protein of interest. The measured fluorescent intensities of cells were normalized to PS averages within each set and then pooled.

Statistical Analysis

Unless otherwise specified, each experiment was repeated at least 3 times, and each independent experiment was performed with SZC that were pooled from multiple bovine legs. Data collected from each set of experiments were aggregated. Statistical analysis wase performed using GraphPad Prism (GraphPad Software Inc, Boston, MA). The aggregated data were examined for outliers using the ROUT method.³⁷ To examine differences between 2 groups, we used an unpaired t test. For greater than 3 groups, an analysis of variance (ANOVA) was used to determine whether differences existed between group means, followed by planned comparisons using the Bartlett correction method to detect differences between groups of interest. Group comparisons for each set of gene expression data are shown in Supplementary Table S3. Data are presented as mean ± standard deviation.

Results

Culture of P0 SZC on CD-ECM Enhances Cellular Attachment

To characterize the degree of decellularization, CD-ECM was assessed through immunofluorescence and DNA quantification. Immunofluorescent staining showed no detectable cells or nuclear material (Suppl. Fig. S1A). Furthermore, CD-ECM had a residual DNA content of 0.25 ± 0.03 μg/ml, compared with CD-ECM with cells having a DNA content is 17.59 ± 1.52 μg/ml (Suppl. Fig. S1B). This indicates the CD-ECM was effectively decellularized.

To evaluate whether seeding primary SZC on CD-ECM increases attachment, we seeded equivalent numbers of P0 SZC on either PS or CD-ECM. Light microscopy reveals that there is greater SZC coverage on CD-ECM as compared with the PS ( Fig. 1A ). We calculated the percentage area covered by cells in our obtained microscopy images and found that SZC on CD-ECM occupied a greater area (5.9% ± 1.0%) as compared with SZC on PS (1.9% ± 0.5%) ( Fig. 1B ). In addition, to compare the amount of SZC that are attached on CD-ECM compared with PS, we extracted and quantified DNA content as a proxy of cell number. We found that there was a greater amount of DNA extracted from CD-ECM dishes than PS ( Fig. 1C ). These results indicate that seeding P0 SZC on CD-ECM increases the proportion of SZC on PS.

Figure 1.

Primary SZC attachment on PS and CD-ECM. (A) Light microscopy images and quantified (B) percent area covered of P0 SZC attached to PS and CD-ECM (CM) after 24 hours. (C) Percent Attachment of SZC seeded on PS and CD-ECM after 24 hours. **P < 0.01, ***P < 0.001.

Expansion of SZC on CD-ECM Reduces Time to Confluency

Next, we aimed to determine the length of time it takes for freshly isolated SZC to expand to confluency (80%-90% cell coverage) on CD-ECM from the primary (P0) stage to P1, and then from P1 cells to P2. Confluency was examined by calculating cellular coverage on light microscopy images at each day of culture. P0 SZC reached confluency on days 13 and 8 on PS and CD-ECM culture flasks, respectively. On day 8, when SZC on CD-ECM reached confluency, the SZC on PS were found to be 18.7% ± 2.0% confluent ( Fig. 2A and B ). Expansion of SZC from P1 to P2 results in SZC reaching confluency by 7 days on CD-ECM as compared with 9 days on PS. On day 7, when SZC on CD-ECM reached confluency, SZC on PS were found to be 51.9% ± 7.7% confluent ( Fig. 2C and D ). These results demonstrate that SZC on CD-ECM reach confluency faster as compared with SZC on PS.

Figure 2.

Proliferation of SZC on PS and CD-ECM from P0-P1 and P1-P2. (A, C) Percent area covered of SZC passaged on PS or CD-ECM each day as cell progress from (A, B) P0 to P1 and (C, D) P1 to P2. (B, D) Representative light microscopy images at Day 8 and Day 7, respectively. **P < 0.01, ***P < 0.001.

Expansion of SZC on CD-ECM Represses the Expression of Dedifferentiation Molecule mRNA Levels

To evaluate the effect of monolayer expansion on SZC dedifferentiation, we compared the mRNA levels of SZC expanded on either PS or CD-ECM at each passage. With regard to chondrogenic expression ( Fig. 3A ), we found no differences between PS and CD-ECM ACAN mRNA levels at P0 and P1. However, SZC on CD-ECM had higher ACAN mRNA levels at P2 compared with PS. Superficial zone chondrocytes expanded on PS and CD-ECM showed significant decreases in ACAN mRNA levels between P0 and P2. At P0, COL2 mRNA levels of SZC on PS were higher than those on CD-ECM. There is no significant difference in COL2 mRNA levels between SZC expanded on PS and CD-ECM at P2, while both showed significant decreases between P0 and P2. SRY-Box Transcription Factor 9 (SOX9), a known transcriptional activator of ACAN and COL2,^38,39 mRNA levels were higher in SZC passaged on CD-ECM at P0 and P2.

Figure 3.

Gene expression analysis of SZC passaged on PS and CD-ECM at P0, P1, and P2. Real-time RT-PCR of (A) chondrogenic (ACAN, COL2, and SOX9), (B) SZ-specific (PRG4 and CLU), (C) dedifferentiation (COL1, αSMA, and TNC), and (D) proliferation (KI67 and CCND1) mRNA levels of SZC passaged on PS and CD-ECM (CM) at P0, P1, and P2. Expressed as a percent of P0 PS. *P < 0.05, **P < 0.01, ***P < 0.001.

We found that mRNA levels for SZ-specific PRG4 ( Fig. 3B ) are higher at P0 for SZC seeded on CD-ECM compared with PS. By P1, PRG4 mRNA levels in SZC cultured on CD-ECM are lower as compared with P0. SZ-specific CLU mRNA levels increased in SZC passaged on CD-ECM by P2, where levels were significantly higher than PS.

To determine the effects of passaging on dedifferentiation markers we examined COL1, αSMA, and TNC mRNA levels ( Fig. 3C ) throughout passage. At P0 there are no differences in COL1 and αSMA mRNA levels between PS and CD-ECM. Throughout passage, there are increases in COL1 and αSMA mRNA levels between P0, P1, and P2 on PS, while there are no increases throughout passage on CD-ECM. Similar trends are seen in mRNA levels for TNC. At P0 there is no difference in TNC mRNA levels in SZC on CD-ECM or PS. Throughout passaging, there is an increase in TNC mRNA levels between P0 and P1 on PS. For CD-ECM there are no differences in mRNA levels throughout passaged. By P2, there is a significant decrease in mRNA levels on PS, showing no significant difference between PS and CD-ECM at P2.

Since SZC passaged on CD-ECM reached confluency faster than SZC on PS, we also examined the mRNA levels for the proliferation markers KI67 and CCND1 mRNA levels ( Fig. 3D ). We found no differences throughout passaging on KI67 and CCND1 mRNA levels of SZC on both PS and CD-ECM. While elevated, mRNA levels were not significantly higher between SZC on PS and CD-ECM at P0 and P1 but were significantly higher at P2. While passaging SZC on CD-ECM does not prevent the loss of chondrogenic and SZ-specific mRNA levels, it represses dedifferentiation marker mRNA levels in passaged cells.

Passaging SZC on CD-ECM Leads to Smaller Cells with Greater G-/F-Actin

Increases in chondrocyte size and actin polymerization are positive regulators of dedifferentiation gene expression.^{13
-15,17,18,22,23} Since passaging on CD-ECM led to substantial repression of the dedifferentiation molecules (i.e. αSMA and COL1), we sought to determine if culturing on CD-ECM represses cellular spreading and actin polymerization. We found there to be no differences in cell area and circularity ( Fig. 4A-C ) of P0 SZC cultured on PS and CD-ECM. Between P0 and P2 SZC on PS and CD-ECM increased in area. Interestingly, at P2, SZC on CD-ECM are smaller compared with PS. We found there to be no differences in circularity in P0 or P2 cells cultured on PS versus CD-ECM.

Figure 4.

Morphology at P0 and P2 and actin organization at P2 of SZC passaged of PS and CD-ECM. (A) Light microscopy images of SZC on PS and CD-ECM at P0 and P2. (B) Area and (C) Circularity of P0 and P2 SZC on PS and CD-ECM (CM). Confocal microscopy showing (D) F-Actin (red), G-Actin (green), and Nuclei (Blue) of P2 SZC on PS and CD-ECM. (E) G/F-Actin fluorescent intensity ratio. Capillary Western immunoassay (F) psuedoblot and corresponding (G) plot of Triton-fractionated samples for separation of G/F-Actin of P2 SZC on PS and CD-ECM. **P < 0.01, ***P < 0.001.

To determine if the reduced area corresponds to reduced F-actin, we stained P2 SZC on CD-ECM and PS were stained for F- and G-actin ( Fig. 4D ). Culturing SZC on both PS and CD-ECM led to reorganization of F-actin into stress fibers. We found that P2 SZC on CD-ECM have less F-actin stress fibers ( Fig. 4D ) and a higher proportion G-/F-actin ( Fig. 4E ). To confirm the greater proportion of G-actin in P2 SZC on CD-ECM, we separated Triton-soluble (predominantly G-actin) and Triton-insoluble (predominantly F-actin) fractions from cells and performed WES capillary electrophoresis. We found a higher proportion of G-actin in cells on CD-ECM as compared with PS ( Fig. 4F and G ).

Overall, passaging SZC on CD-ECM limits SZC spreading, the formation of actin stress fibers, and increases the proportion of G-/F-actin which may regulate the gene expression of the SZC.

P2 SZCs from CD-ECM Cultures Deposit Cartilage-like Tissue When Redifferentiation in 3D Scaffold-Free Cultures

P2 SZC from PS expansion culture are capable of redifferentiation and can be stimulated to deposit cartilage-like matrix.²² To determine the capabilities of P2 SZC from CD-ECM culture to redifferentiate and deposit matrix, we seeded P2 SZC from CD-ECM cultures in 3D within agarose molds and maintained cells in culture media that promotes redifferentiation.^22,23 We found there to be no differences in ACAN mRNA levels of P2 SZC derived from CD-ECM and PS at days 10 and 21 of redifferentiation culture. In addition, while P2 SZC from CD-ECM had higher COL2 mRNA levels as compared with P2 SZC derived from PS culture at 10 days of culture there were no differences in COL2 mRNA levels by day 21. We also determined that SOX9 mRNA levels are higher in P2 SZC from PS on day 10 of redifferentiation. However, by day 21 there is no difference in mRNA levels between P2 SZC derived from CD-ECM and PS cultures.

At day 10 of redifferentiation culture, fibroblastic markers COL1, αSMA, and TNC mRNA levels are lower in P2 SZC from CD-ECM compared with PS. Although by day 21 of redifferentiation culture both P2 SZC from PS and CD-ECM have similar COL1, αSMA, and TNC mRNA levels. By day 21 of redifferentiation culture, P2 SZC from PS and CD-ECM have similar chondrogenic and fibroblastic matrix marker mRNA levels.

Next, we evaluated the matrix tissues generated by P2 SZC derived from CD-ECM culture. Similar to P2 cells derived from PS culture, H&E staining ( Fig. 5B ) shows production of extracellular matrix by P2 cells from CD-ECM on day 21. Toluidine Blue staining ( Fig. 5B ) of histological sections shows similar staining for accumulation of proteoglycans in both PS and CD-ECM derived tissues. Similarly, picrosirius red staining ( Fig. 5B ) shows similar staining for collagen accumulation in both tissues. To examine the accumulation of specific chondrogenic ( Fig. 5C ) and fibroblastic ( Fig. 5D ) matrix proteins, day 21 tissue sections were stained for ACAN, COL2, and COL1. In terms of chondrogenic matrix expression, tissues derived from SZC passaged on CD-ECM have higher staining intensity for ACAN compared with PS ( Fig. 5C and E ). PS and CD-ECM derived tissues had similar staining intensity for COL2. To confirm the formed tissues had limited fibroblast matrix accumulation and any difference between redifferentiated P2 PS and CD-ECM SZC, tissues were stained for COL1 ( Fig. 5D ). Both tissues have limited COL1 staining, while having similar intensity ( Fig. 5E ). Similar to P2 SZC passaged on PS, P2 SZC from CD-ECM culture are also capable of forming cartilage-like matrix.

Figure 5.

Characterization of tissues formed by P2 SZC redifferentiated in 3D scaffold-free culture following expansion on PS and CD-ECM at day 10 and 21. (A) Real-time RT-PCR of chondrogenic (ACAN, COL2, and SOX9) and fibroblastic (COL1, TNC, and αSMA), matrix marker mRNA levels of redifferentiated P2 SZC passaged on PS and CD-ECM (CM). Expressed as a percent of P0 Day 10 (B) H&E, Toluidine Blue, and Picrosirius Red staining of tissues derived from passaged on PS and CD-ECM. Confocal microscopy images showing matrix accumulation of (C) chondrogenic proteins ACAN and COL2 and (D) fibroblastic matrix protein COL1. Specific proteins are shown in grayscale, and tissues were counterstained with Hoechst for Nuclei (Blue). (E) Quantification of ACAN, COL2, and COL1 mean fluorescent intensity (F.I.). *P < 0.05, **P < 0.01, ***P < 0.001.

P2 SZC Derived from CD-ECM in 3D Scaffold-Free Redifferentiation Cultures Express Higher PRG4

To evaluate the ability of SZC passaged on PS and CD-ECM to form SZ-like tissues after redifferentiation, real-time reverse transcription PCR and fluorescence microscopy were used to examine SZ-specific expression. Tissues formed by P2 SZC passaged on CD-ECM had significantly higher PRG4 mRNA levels at day 21 of culture compared with PS ( Fig. 6A ). To examine PRG4 expression and localization, tissues were stained for PRG4 ( Fig. 6B ). While both tissues derived from SZC passaged on PS and CD-ECM express PRG4, which localizes close to tissue surfaces, tissues from CD-ECM derived SZC have higher fluorescent intensity ( Fig. 6C ). While both SZC passaged on PS and CD-ECM produce chondrogenically favorable tissue, CD-ECM derived tissues contain higher levels of PRG4.

Figure 6.

PRG4 expression of 3D scaffold-free tissues derived from P2 SZC passaged on PS and CD-ECM at day 10 and 21. (A) Real-time RT-PCR of SZ-specific PRG4 mRNA levels of PS and CD-ECM (CM) tissues at day 10 and day 21 of culture. mRNA levels are expressed as a percent of PS Day 10 mRNA levels. (B) Confocal microscopy images showing PRG4 accumulation in PS and CD-ECM derived tissues at day 21. PRG4 is shown in grayscale and tissues were counterstained with Hoechst for Nuclei (Blue). (C) Quantification of PRG4 mean fluorescent intensity (F.I.). *P < 0.05, ***P < 0.001.

Discussion

This study determined that the culture of SZC on CD-ECM improves SZC expansion through increased primary SZC cell attachment, decreased time for SZC to reach confluency, and a reduction in the expression of differentiation genes in P2 SZC. The expansion of SZC on CD-ECM led to elevated PRG4 expression when P2 SZC were redifferentiated to form bioengineered tissue. Overall, these findings lend support to our hypotheses that CD-ECM will promote attachment and proliferation of SZC. Passaged SZC derived from CD-ECM culture are less dedifferentiated and have a superior capacity to form cartilage tissues that express PRG4.

Culturing SZC on CD-ECM enhances SZC attachment. Previous studies have identified poor primary SZC attachment onto polystyrene.^22,25 The poor attachment could lead to a small portion of SZC left in culture when passaging full-thickness chondrocytes, which contain deeper zone cells which attach and proliferate at a higher degree than SZC. Since only SZC can re-express PRG4, the poor primary attachment of SZC may limit the ability to form a proper SZ in bioengineered cartilage using passaged full-thickness chondrocytes.^20
-22 Here, we found that seeding primary SZC on CD-ECM increases cellular attachment. The superior cellular attachment on CD-ECM may be due to increased interaction between cells and ECM molecules. While the mechanism(s) promoting SZC on CD-ECM are not known, chondrocytes express several integrins to enable attachment to extracellular matrix proteins such as fibronectin (α5β1), COLII and COLVI (α1β1, α2β1, α10β1), laminin (α6β1), osteopontin (αVβ3).^40
-42 Our mass spectrometry analysis revealed ECM proteins fibronectin-1 and collagen type VI to be the top hits in CD-ECM (Suppl. Fig. S4), which may facilitate enhanced cell attachment and signaling through integrin-mediated pathways that support maintenance of the SZC phenotype during expansion. In the current study we did not elucidate the mechanism leading to increased attachment, however, future studies to identify how CD-ECM increases primary SZC attachment could focus on determining if increased binding of SZC is due to individual or a combination of specific ECM molecules. Nonetheless, the ability to increase the primary SZC when seeding on CD-ECM could aid in solving the limitations of SZC expansion on PS.

SZC on CD-ECM significantly reduces the time cells to reach confluency. An additional challenge in obtaining an adequate SZC population after passaging is their slow proliferation rate compared with other zonal chondrocytes.^22,25 One potential mechanism leading to shorter time to confluency for SZC passaged on CD-ECM may be the increased attachment to CD-ECM. With increased attachment, there is a larger initial cell population that can proliferate leading to reduced time to reach confluency. Alternatively, we also showed elevated mRNA levels of KI67 and CCND1 in cells cultured on CD-ECM, which may indicate that the CD-ECM is stimulating increased proliferation of the SZC. Future studies are required to determine the mechanism behind the reduced time for SZC on CD-ECM to reach confluency. Nonetheless, passaging SZC on CD-ECM may help in overcoming the challenge of slow proliferation exhibited by SZC passaged on PS.

Expansion of SZC to P2 on CD-ECM repressed dedifferentiation. Expansion of SZC on PS led to a loss of chondrogenic expression and an increase in fibroblast matrix and contractile molecule expression, consistent with previous studies that investigated the dedifferentiation of full-thickness chondrocytes by expansion on PS.^{13
-15,17,18,22,23} While culture on CD-ECM did not prevent the reduction of chondrogenic mRNA levels, culture on CD-ECM represses fibroblastic matrix and contractile molecule mRNA levels in passaged SZC. A previous study similarly demonstrated a repression of fibroblast matrix mRNA levels during full-thickness chondrocyte expansion on CD-ECM.²⁸ The actin cytoskeleton is a potent regulator of the dedifferentiated phenotype in passaged chondrocytes. During monolayer expansion chondrocytes form F-actin stress fibers.¹³ In this process, the proportion of monomeric G-actin is reduced, which upregulates the expression of fibroblastic matrix and contractile genes by increasing the nuclear proportion of a G-actin binding transcription factor, myocardin-related transcription factor.¹⁵ In the present study, we found that culturing SZC on CD-ECM limited stress fiber formation and increased the proportion of G/F-actin, which led to a reduction in fibroblastic matrix and contractile expression. Therefore, CD-ECM aids in repressing dedifferentiation, potentially signaling through actin-related pathways.

Our study demonstrates that both SZC passaged on PS and CD-ECM can be redifferentiated in 3D scaffold-free culture to produce tissues expressing cartilage matrix proteins and SZ-specific PRG4. Intriguingly, tissues derived from SZC passaged on CD-ECM had higher expression of PRG4. While the mechanism behind the increased PRG4 expression warrants future studies to aid in generating adequate PRG4 production in bioengineered tissues. Interestingly, the PRG4 expressed in both the PS and CD-ECM derived tissues is localized to the tissue surface, similar to the articular surface of native articular cartilage. While the mechanism behind the localization of PRG4 in the 3D redifferentiation scaffold-free tissues in unknown, there a several factors that may contributing. One possible contributing factor could be mechanical stimulation, which has been shown to increase PRG4 expression.⁴³ In our 3D redifferentiation culture the SZC on the bottom could potentially be sensing mechanical forces generated via attachment to the PS, leading to increased PRG4 production. Similarly, increased oxygen access has been shown to stimulate PRG4 expression,⁴⁴ which may contribute to increased PRG4 expression on the top surface of our tissues due to the increased availability of oxygen. A further understanding of the underlying mechanism of PRG4 localization to the surface of tissue may allow for better generation of a native-like SZ in bioengineered cartilage.

Our present study showed an enhanced ability to redifferentiate bovine SZC that were expanded on CD-ECM. The ability to passage and redifferentiate human SZC on CD-ECM was not examined, though the study provides a conceptual framework to apply to the passaging and redifferentiation of human SZC. While species-specific differences must be taken into account, our findings suggest that CD-ECM could similarly benefit human SZC by promoting cell attachment, maintaining superficial zone identity, and enhancing redifferentiation. Future studies will be necessary to validate this approach using primary human SZC, and doing so will help determine the translational potential of CD-ECM-based cell expansion systems for cartilage repair strategies. Our findings show that SZC can be expanded to P2 on CD-ECM with sufficient yields for tissue engineering applications while retaining key superficial zone characteristics. Although higher passages (P3, P4) were not assessed, chondrocytes typically lose phenotype with extended passaging.¹⁴ Further studies are needed to evaluate whether CD-ECM can preserve SZC identity beyond P2, and their capacity to redifferentiate to form SZ-like tissue.

Furthermore, this study does not examine the difference in expansion and redifferentiation of chondrocytes harvested from healthy or diseased tissue. The SZ of native articular cartilage is the first effected in the progression of OA.⁷ These cells also experience a shift from their normal metabolic environment, experiencing an increase in inflammatory cytokines and degrative enzymes.⁴⁵ Currently it is not known whether these cells have a similar capacity to be expanded and redifferentiated to SZC harvested from healthy tissue. Future studies should address the capability of chondrocytes harvested from OA tissue to be passaged and used for bioengineering repair tissue.

Our study focuses on optimizing the culture of SZC and does not examine the expansion of middle and deep zone chondrocytes on CD-ECM. Further studies would be required to determine the effects of passaging other zones on CD-ECM. In addition, while this study demonstrates the benefits of CD-ECM in maintaining SZC phenotype during expansion and redifferentiation, future work should explore the behavior of these cells in more complex systems. Co-culture with deeper zone chondrocytes or integration into cartilage organoids may better reflect in vivo environments and cell to cell interactions critical for layered cartilage regeneration. Although SZC are specialized for the superficial cartilage environment, it remains unclear whether they can adapt to the condition’s characteristic of deeper cartilage layers, such as low oxygen tension and elevated osmolarity. These factors are well-established chondro-inductive cues that support matrix production and phenotypic stability in deeper zone chondrocytes.^46,47 Nonetheless, the use of CD-ECM could be effective in repairing SZ tissue.

This study demonstrated that passaging SZC on CD-ECM improves SZC expansion through increased primary cell attachment, decreased time to reach confluency, and the limiting of differentiation. In addition, SZC passaged on CD-ECM can be redifferentiated to form bioengineered tissue that expresses higher levels of PRG4 than traditional culture on PS. CD-ECM may help in providing a strategy to aid in overcoming the challenges posed in obtaining and passaging SZC on uncoated PS. The ability to achieve an adequate population of SZC may prove crucial in generating bioengineered cartilage with native-like zonal cellular arrangement and expression.

Supplemental Material

sj-pdf-1-car-10.1177_19476035251369735 – Supplemental material for Enhanced Superficial Zone Chondrocyte Expansion and Redifferentiation by Culture on Chondrocyte-Derived Decellularized Matrices

Supplemental material, sj-pdf-1-car-10.1177_19476035251369735 for Enhanced Superficial Zone Chondrocyte Expansion and Redifferentiation by Culture on Chondrocyte-Derived Decellularized Matrices by Thomas J. Manzoni, Anh Ho, Lilly Smull, Valarie C. West, Jeffery D.V. Waters, Karina Lemus, James Adams, Alvin W. Su and Justin Parreno in CARTILAGE

Footnotes

ORCID iDs

Thomas J. Manzoni

Alvin W. Su

Justin Parreno

Ethical Considerations

Not applicable

Acknowledgments and Funding

The author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: This work was supported by a JRF Ortho (Englewood, CO, USA) Research Grant to JP and AWS, an American Orthopedic Society for Sports Medicine Steven P. Arnoczky Young Investigator Grant to AWS and JP, and a University of Delaware Chemistry and Biology Interface NIH fellowship (5T32GM133395-03) to TJM. In addition, support was provided by the Delaware INBRE program from the National institute of General Medical Sciences—NIGMS (P20 GM103446), Delaware Center for Musculoskeletal Research from the National Institutes of Health General Medical Sciences- NGIMS (P20 GM139760). We would also like to acknowledge Bob Hutchens and StemBioSys for support.

Declaration of Conflicting Interests

The author(s) declared the following potential conflicts of interest with respect to the research, authorship, and/or publication of this article: Thomas J. Manzoni (None), Anh Ho (None), Lilly Smull (None), Valarie C. West (None), Jeffery D. V. Waters (None), Karina Lemus (StemBioSys), James Adams (StemBioSys), Alvin W. Su (None), Justin Parreno (None).

Supplementary material for this article is available on the Cartilage website at .

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Supplementary Material

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