Enhancement of chondrogenic differentiation in ATDC5 cells using GFOGER-modified peptide nanofiber scaffold

Introduction

Articular cartilage is a complicated tissue that covers the surface of bones in synovial joints [1]. It distributes and transfers loads to the subchondral bone while allowing smooth articulation with minimal friction. Additionally, articular cartilage has a limited repair ability after injury below because of its avascular and aneural features. Microfracture and autologous chondrocyte implantation are two contemporary therapies that quicken articular cartilage tissue regeneration. However, long-term advantages are challenging to attain [2]. When the regenerated tissue finally fails due to the lack of original articular cartilage biomechanical characteristics, further treatment, such as complete joint arthroplasty, is required [3]. Because of this, there has been an upsurge in interest in developing bioengineered structures to foster the bioinspired environment necessary to assist the regeneration of destroyed native tissue [4]. The capacity to construct tissues with composition and architecture comparable to native articular cartilage holds considerable promise for restoring the precise biomechanical and functional qualities required for prolonged usage.

The interactions between cells and extracellular matrix (ECM) play a crucial role in the differentiation and formation of tissues. Integrin-mediated fibronectin attachment is essential for limb bud cell condensation, and certain fibronectin isoforms regulate the degree of condensation [5], [6], [7]. Type II collagen interactions can also stimulate mesenchymal stem cell chondrogenesis in vitro [8]. Further investigation is required to fully comprehend the intricate mechanisms of cell-matrix interactions involved in chondrogenesis. However, it is acknowledged that the extracellular matrix (ECM) plays a pivotal role in driving this process by providing important signals. One approach for modifying the interactions between cells and matrices in a laboratory setting involves creating artificial matrices that contain ligands. Research has demonstrated that incorporating peptides containing the integrin-based sequence arginine-glycine-aspartic acid (RGD) into non-reactive materials improves cell attachment, extends the lifespan of mesenchymal stem cells [MSC], and encourages the development of bone-forming cells [8].

Collagens are fibrillar proteins in the ECM linked to cell functions such as proliferation, differentiation, and cell–cell or cell–ECM communication. Several studies have found collagen hydrogels to be inductive for chondrogenesis [9, 10]. However, the synthetic collagen may be immunogenic and vary from batch to batch. Because of concerns related to native collagen or naturally derived collagen products, several studies employed collagen-mimicking hydrogels to recapitulate native ECM.

Several integrins bind to bioactive collagen patterns and initiate intracellular cytoplasmic signaling cascades. It is generally known that important biological processes, such as tissue formation, regeneration, and homeostasis, depend on integrin-collagen interaction [11]. Therefore, it is crucial to investigate and evaluate the function and physical properties of collagen-like biomaterials that mimic the structure and features of collagen. Unfortunately, the biological applicability of research on collagen-mimicking sequences is restricted primarily due to their small size, which is not comparable to native collagen. Several researchers have attempted to create artificial collagen fibers with the same qualities as native collagen fibers by chemical and self-assembly processes. Recently published research has focused on the production of natural collagen fibers.

Moreover, current studies demonstrate the effective production of artificial collagen fibers with certain biophysical qualities like native collagen, closing the gap between their biophysical properties. Several techniques have been used to produce triple helical fibers at the micrometer scale, including a π-stacking approach, metal-assisted self-assembly for obtaining long fibers, and a study that developed small collagen segments where the three strands were maintained in a staggered array using disulfide bonds [12, 13]. Nevertheless, further effort is required to close this gap, particularly regarding incorporating biological features. The production of synthetic collagen fibers with structural and bio-functional features may have major potential uses in tissue regeneration.

Through a bottom-up approach to self-assembly using PA, we aim to replicate collagen-based peptides to create synthetic collagen fibers with the same physical and bio-functional properties as native collagen in this study. It has been shown that the molecular self-assembly method offers flexibility for concurrently manipulating nanostructure and chemical functionality. Undifferentiated chondrogenic ATDC5 cells were used to observe the effect of peptide-based collagen-mimetic scaffold on in vitro chondrogenic differentiation [14, 15]. ATDC5 cells are a well-established in vitro model for chondrogenic differentiation studies due to their responsiveness to chondrogenic stimuli and ability to undergo morphological and biochemical changes similar to those seen in chondrocytes [15]. Chondrogenic differentiation is a complex biological process that involves activating several signaling pathways and transcription factors, such as BMP signaling, Sox9, and Runx2. Upon exposure to chondrogenic stimuli via TGF-β1 or BMP-2, ATDC5 cells exhibit an increase in the expression of extracellular matrix proteins like aggrecan and collagen II and in the expression of chondrogenic markers like Col10a1 and Sox9 [16]. Researchers have utilized this system to examine the molecular mechanisms involved in the process of chondrogenic differentiation and to assess the effectiveness of various therapeutic agents for this condition [17]. The use of ATDC5 cells has provided valuable insights into the molecular processes involved in chondrogenic differentiation and can potentially contribute to developing novel therapies for cartilage-related diseases. Overall, this study has scope to explain the effect of the peptide network in which the GFOGER sequence is integrated to mimic collagen fibrils on cartilage differentiation, especially at the gene level.

Materials and methods

All materials, including amino acids and HBTU for PA production from NovaBiochem, lauric acid DIEA from Merck, and cell consumables from Biological Industries (Kibbutz Beit Haemek) and cell culture consumables from Nest Scientific USA Inc, were purchased for this study. ATDC5 cells were acquired by donation from Hacettepe University.

The synthesis of PA molecules was carried out using a solid-phase peptide synthesis (SPPS) method based on fluorenylmethyloxycarbonyl (Fmoc) chemistry. The Fmoc-protected amino acids were attached to a preloaded Wang resin via a peptide bond formation. After each coupling step, the Fmoc protecting group was removed using a solution of 20 % piperidine in dimethylformamide (DMF), exposing the free amine group for the next coupling reaction. The side chain protecting groups were also removed selectively to allow for specific peptide functionalization [18]. The steps for PA synthesis were applied as explained in detail in the literature [18].

The PA molecules were purified after synthesizing through a reverse-phase HPLC system [19]. PAs’ molecular weight and purity were determined using an Agilent LC–MS system with ESI-MS. The purity of the PAs was measured at 220 nm with respect to the peptide content. To produce 1 % aqueous solutions of all PA molecules at pH 7.4, diluted HCl or NaOH was used and combined in a 1:1 (v/v) ratio. The synthesized nanofibers were dried in ethanol at increasing concentrations for the first 10 min and then dried in a Tourismis Autosamdri critical dryer. The samples were then coated with 3 nm of Au/Pd and analyzed using an FEI Quantum 200 SEM in a high vacuum [20].

ATDC5 cells are a mouse chondrogenic cell line commonly used as a model for studying chondrogenic differentiation. The cells were maintained as monolayer cultures on tissue culture plates (TCP) under conventional growth conditions, which involved incubation at 37 °C with 5 % CO₂. The culture medium used was a mixture of DMEM and Ham’s F12 medium in a 1:1 ratio, supplemented with 5 % fetal bovine serum (FBS), 10 mg/L holo-transferrin, and 3 × 10⁻⁸ M sodium selenite. FBS provides essential nutrients and growth factors for cell growth and division, while holo-transferrin and sodium selenite regulate cell metabolism.

Ten mg/L of insulin was added to the culture medium to induce chondrogenic differentiation. Adding insulin to the culture medium is likely intended to promote the differentiation of ATDC5 cells into chondrocytes, which eventually form the cartilage tissue.

Results

Formation collagen-mimetic nanofibers

PAs refer to peptide amphiphiles with a chain of alkyl groups at one end. By adding amino acids with an electrical charge, it is easy to make these molecules dissolve in water, and they assemble themselves into nanofibers through a process known as self-assembly. This process can be triggered by pH shifts, ionic substances, and molecules with opposite electrical charges [22]. In an aqueous environment, fibers create microscale 3D networks and macroscale gels that contain water [23]. By incorporating physiologically functional amino acid sequences into the PA sequence, the functionality of the extracellular matrix may be reproduced, and an environment conducive to cell proliferation or differentiation can be created. In this study, we employed a collagen-mimicking functional GFOGER sequence, which binds to the cells through α1β1 and α2β1 integrins. Specifically, collagen-integrin 1 interactions are crucial for chondrocyte aggregation and phenotypic maintenance in suspension culture, and the GFOGER region of the collagen 1 chain corresponds to residues 502–507. Electrostatic and hydrophobic interactions between oppositely charged PA molecules formed a nanofibrous network at physiological pH [24].

This study produced PA hydrogel by combining positively charged K-PA as a charge neutralizer to form a fiber. The synthesized peptides’ results from liquid chromatography (LC) and mass spectrometry (MS) showed that their molecular weights matched the theoretical values, demonstrating the effectiveness of the synthesis (Figure 1B–E). The pureness of the synthesized peptides was demonstrated by a single, dominating peak in the LC data. The nanofiber network formed by mixing GFOGER-PA and K-PA was visualized using scanning electron microscopy. The nanofiber structure had structural characteristics with the natural extracellular matrix, including matrix fibril width and porosity (Figure 1D).

Figure 1:

(A) Chemical structures of PA molecules. MS spectrum of (B) GFOGER-PA and (C) K-PA. LC chromatogram of (D) GFOGER-PA and (E) K-PA. (F) Scanning electron microscope micrograph of indicated nanofibers.

Viability analysis of ATDC5 on collagen-mimetic nanofibers

Experiments on cell survival and cell adhesion were used to assess the early cellular responses of ATDC5 cells to peptide nanofibers. The survival of cells was evaluated on days 1, 2, and 3. Based on our results, the survivability rates on Day 1 and Day 3 did not differ statistically between the cell culture dish and the peptide nanofiber group. However, the cell proliferation rate cultivated on TCP on Day 2 was statistically greater than the peptide nanofiber group. When cells are grown on a peptide nanofiber scaffold, their adhesion, migration, and proliferation conditions are affected due to high signals received from the environment. Cells exposed to the GFOGER signal and the negative signal in the scaffold may activate intracellular pathways and differentiate. Thus, cellular responses like proliferation and adhesion are anticipated to differ from those of the control group.

Moreover, the delayed proliferation response observed on Day 2 is compensated on Day 3. Consequently, it can be concluded that peptide nanofibers have no negative effect on both the metabolic reactions of cells and the proliferation rate (Figure 2A). Using calcein AM staining showed that ATDC5s were attaching to the peptide coatings and altering shape after being seeded (Figure 2C). When comparing the number of cells that adhered to the peptide nanofiber scaffolds to those that adhered to the traditional tissue culture plates (TCP), there was little difference between the two groups (averages of 157 and 189 % for peptide nanofibers vs. averages of 147 and 173 % for TCP at 2 and 5 h, respectively). Three hours into the culture, both groups exhibited an enhanced rate of cell attachment, but there was no notable distinction between the two groups in terms of cell attachment. The usage of calcein AM staining revealed that the peptide nanofibers could support cell attachment, as evidenced by changes in the shape of the cells after they were seeded onto the nanofibers (Figure 2C).

Figure 2:

(A) Bio viability analysis of ATDC5 for day 1, 2 and 3. (B) Representative morphology of ATDC5 on GFOGER/K-PA and TCP. (C) Adhesion of ATDC5 on GFOGER/K-PA and TCP. (D) Relative adhesion quantitation of ATDC5 cells at 2 and 5h on GFOGER/K-PA and TCP.

At both 2 and 5-h time points, there was no significant difference in the quantity of cells attached to the peptide nanofiber scaffolds compared to those attached to the TCP (with no statistically significant difference between the two groups). This data indicates that fiber networks facilitate cell adhesion and do not impede cell attachment (Figure 2C). When cellular morphology was followed, it was seen that cellular shape was quite different between the two groups. For example, cells on peptide nanofiber scaffold have gained a round shape and cellular condensation, whereas cells on TCP keep spindle shape, which does not exhibit aggregation behavior. Treatment with insulin induces a process of cellular condensation in ATDC5 cells, causing subsequent chondrogenic differentiation, characterized by the production of proteoglycans and expression of type II collagen [15]. These changes led to a result that cells respond to an inductive culture environment because cells showed aggregate formation on GFOGER/K-PA.

Chondrogenic differentiation analysis

In the differentiation study, ATDC5 was cultured on a peptide scaffold and on TCP in media supplemented with or without insulin. It has been shown in the literature that ATDC5 cells differentiate from prechondrogenic cells to form cartilage cells in the presence of insulin [15]. Therefore, the ATDC5 cells were cultured in a medium with or without insulin to examine the cells’ differentiation responses. RNAs were collected for the gene expression analysis of cultured cells, and the expression rates of Collagen II and Sox9 genes, which are important markers for cartilage differentiation, were examined. Accordingly, when cells were cultured on GFOGER-PA/K-PA in a media without insulin, they significantly increased Collagen II expression on Day 5 (fold change:4.73) and Day 10 (fold change:7.76) relative to TCP (Figure 3A and B). The same trend was also valid for cells grown in media with insulin (D5 fold change:5,64 and D10 fold change:8,42).

Figure 3:

(A, B) Collagen II expression of ATDC5 on indicated surfaces at day 5 and 10 in medium either supplemented with (A) or without insulin (B). (C) Sox9 expression levels of ATDC5 cell on day 5. The level of expression for every gene was adjusted based on the uncoated TCP samples where GAPDH was used as an internal control. The values shown indicate the average ± SEM, with a sample size of 3 (^***p<0.0001, ^**p<0.01, *p<0.05). (D) Representative morphology images of ATDC5 on indicated surfaces on day 5 and 10 in medium either supplemented with or without insulin.

Similarly, the expression of the Sox9 gene, which is an important transcription factor for cartilage differentiation, was also screened under the same conditions. Considering the rates of Sox9 expression on Day 5, when the cells were cultured in a medium without insulin, the cells on GFOGER/K-PA showed a 3.38-fold difference, while the cells cultured on TCP showed a 2.18-fold difference. When cells were cultured in insulin, the fold ratios of the Sox9 gene were 6.14 and 2.85 for GFOGER/K-PA and TCP, respectively. It is important to note that Sox9 is an early marker for chondrogenic differentiation. Therefore, additional Sox9 expression data on Day 10 was deemed unnecessary for the scope of this study, which aims to elucidate the rapid and precise differentiation pathway facilitated by the GFOGER-PA/K-PA scaffold (Figure 3C). Considering the increase in both gene expression rates, the cells entered the cartilage differentiation pathway independent of insulin presence on the peptide nanofiber. They showed a higher expression rate than the cells on TCP. Simultaneously, the enhancing effect of insulin was observed when the cells of both experimental groups were cultivated in the presence of insulin. The cells increased the expression of both genes on the 5th and 10th days more than in the non-insulin state. In the literature, ATDC5 cells grown on TCP require 21 days in the presence of insulin to differentiate into cartilage [15]. According to our findings, cartilage differentiation of the cells cultivated on TCP has not yet been completed in 10 days, so cartilage-specific gene expressions on day 10 have not yet been stimulated.

Nevertheless, early differentiation was detected in cells grown on GFOGER-PA/K-PA in both insulin-containing and insulin-free media. Although this result highlights the importance of the GFOGER sequence for cartilage differentiation, the most essential aspect of this work is the presentation of the GFOGER sequence to cells in a structure such as a PA. PA molecules are distinctive in that they offer functional peptides to be integrated into the self-assembling peptide, resulting in a collagen-mimicking, hydrogel-based 3D ECM-like cell network. Because of these benefits, cells are conveyed to a differentiation pathway rapidly and precisely. Furthermore, when cells are cultured on GFOGER- PA/K-PA, they change morphologically, acquire cartilage cell-like morphology, and endochondral formation (Figure 3D).

Discussion

In the present study, implementing collagen-like nanofibers, enhanced by incorporating the GFOGER sequence, provides significant insights into the potentialities of inducing chondrogenic differentiation within ATDC5 cells. By synthesizing peptide amphiphile (PA) nanofibers via self-assembly, we explored an approach marked by its intricacies in alignment with pH changes and ionic dynamics.

An initial assessment of cellular viability revealed noteworthy phenomena, including a slight early-stage proliferation delay, countered by robust growth and metabolism. The underlying mechanics of these observations became clearer as the cells demonstrated remarkable adhesion to the peptide nanofiber structure, reflecting the groundbreaking work of Ha et al. (2023) in osteochondral regeneration using GFOGER-conjugated hydrogels [25].

The morphological transformations of the cells, which were marked by circularity and visible cellular condensation, provided further evidence of the scaffold’s capacity to stimulate intracellular mechanisms leading to differentiation. These findings were augmented by a detailed gene expression analysis that indicated a remarkable elevation in Collagen II and Sox9 gene expression by Day 5 and Day 10. Interestingly, the presence of insulin intensified this cartilage-specific gene manifestation, accelerating the differentiation process. This behavior is in line with the conclusion by Mhanna et al. that the GFOGER peptide outperformed other adhesive peptides such as RGD, enhancing proliferation in degradable PEG gels and fostering a better chondrogenic microenvironment [26].

Collectively, these insights amplify the implications of our research. By successfully implementing collagen-like nanofibers embedded with the GFOGER sequence, we contribute to the cartilage tissue engineering field and lay the groundwork for innovative therapies for cartilage ailments. Our findings, in harmony with previous research, underscore the immense potentialities of such structures in regenerative medicine.

In conclusion, the present study affirms the extraordinary potential of collagen-like nanofiber scaffolds infused with the GFOGER sequence for initiating chondrogenic differentiation in ATDC5 cells. This research opens a promising avenue for treating cartilage-related ailments, particularly when viewed in concert with existing studies. A deeper exploration into the scaffold’s long-term differentiation potential and in vivo efficacy could pave the way for transformative advancements in regenerative medicine’s tissue engineering strategies, catalyzing the development of innovative and effective therapeutic interventions.