Spidroin striped micropattern promotes chondrogenic differentiation of human Wharton’s jelly mesenchymal stem cells

Characterization of human Wharton’s jelly MSCs (hWJ-MSCs)

hWJ-MSCs were characterized based on the Mesenchymal and Tissue Stem Cell Committee of the International Society for Cellular Therapy (ISCT). The features that define MSCs are plastic-adherent when maintained in standard culture conditions, express certain surface molecule markers, and can differentiate to osteoblasts, adipocytes, and chondroblasts in vitro²⁷. The results showed that the cells used in this study were indeed MSCs because they can adhere to plastic, expressing surface molecule markers with > 95% CD105, CD 90, and CD 73 expression, and do not express negative markers (< 2%) such as CD 45, CD 34, CD 14, or IB CD, CD 74 or CD 19, HLA-DR. These results were contrast with MSC marker on fibroblast cells (non-MSC), showed that fibroblast cells only expressed 29.2% of CD 105 and 29.2% of CD90 (Supplementary Data 1, Figure A). hWJ cells can also differentiate into 3 lineages which are chondrogenic, osteogenic, and adipogenic fate that is characterized by the formation of glycosaminoglycan, calcium deposit, and lipid droplets, respectively (Fig. 2). Therefore, the hWJ cells used in this study met the MSC criteria.

Micropattern fabrication using agarose microcontact printing (µCP)

Agarose gel with a striped pattern of 500 µm and 1000 µm was successfully made from agarose 3% (w/v) (Fig. 3A,B). These agarose stamps have a dense consistency but can absorb ink well. The stamp can be stored in PBS at 4 °C and can be used repeatedly during the stamping procedure. The result also showed that the agarose stamp was effective in transferring ink to the substrate as evidenced by the ink pattern that was formed (Fig. 3C,D). Agarose gels have a lower hydrophobicity than other polymers such as PDMS¹⁴, making agarose gel easier to absorb ink and forming a homogenous pattern.

Morphology of hWJ-MSCs cultured on micropattern surface

Figure 4 showed that micropattern facilitates cell adhesion and spreading in a specific area. hWJ-MSCs were grown in the area coated with fibronectin pattern because it serves as an extracellular matrix and can interact with membrane proteins. The morphology of hWJ-MSCs was elongated and normally have cytoplasmic protrusions on the opposite side (Fig. 4A,B). Without micropattern substrate, the cells have a rounded shape that indicates poor adhesion with the surface. The membrane protein, such as integrin, is thought to bind RGD sequences of fibronectin which then leads to strong attachment. Scanning electron microscope (SEM) result showed that the cell shape has already flattened and has several protrusions that interact with the micropatterned substrate (Fig. 4C,D).

Argiope appensa spidroin characterization

Physical properties of spidroin bioink were performed using zeta potential and viscosity. Also, the molecular weight of spidroin solution was estimated according to SDS-page electrophoresis.

Zeta potential is defined as the total charged that a particle has that will determine the ability of a particle to remain disperse or end up agglomerate. The value out of range −30 mV to + 30 mV is considered for particles to have an adequate repulsive force to retain their colloidal stabilities²⁸. Spidroin’s zeta potential has average value −32.8 mV (Fig. 5) therefore show a good particle stability. The bioink shows other physical characteristics that it has viscosity lower than water (0.894 mPa.s). This low viscosity property is according to the SDS page result (Fig. 6) which shows a low kDA result. Low kDA on protein means the protein polymer is a short-chain polymer. This short-chain condition makes the interaction among two or more polymer chains are less strong and easy to be moved by any stirring process.

As shown in Fig. 6, the spidroin was present at around 75 kDa and fibronectin were ranging from 40 to 245 kDa. Compared to another species of Argiope, Argiope aurantia, the extract of spidroin derived from cylindrical gland silk fibers. From these gland, ten major proteins were resolved, with apparent molecular weights ranging from about 13 to over 200 kDa²⁹. Spider silk that we used in this study was derived from spider web, not from the glands. Our result is quite unexpected, since it has different extraction method and source of the spider silk, but the molecular weight is still in the range of 13–200 kDa. These indicating spidroin solution might not degrade when extracted from spider silk.

Argiope appensa spidroin as bioink for µCP micropattern

This research studied the presence of the RGD sequences in spidroin bioink. Based on the ICC result (Fig. 7A), micropattern with spidroin bioink had RGD sequences, shown here as green fluorescence. When the micropattern was overgrown with cells, RGD appeared on the surface of the substrate even after 7 days of culture (Fig. 7B). This result confirmed the research conducted by Barlian et al. (2020) which showed that based on ICC, the 3D scaffold containing the spidroin mixture from Argiope appensa has RGD sequences²¹. In addition, several mechanical testing to characterize spidroin from Argiope appensa has been done, such as by using FTIR spectroscopy analysis²¹. Figure 7A,B proved that spidroin can be used as a bioink for micropattern production and the solubilization process did not damage the RGD sequences. Cell adhesion involves certain cell surface proteins called integrins, and these membrane proteins affect the organization of the cytoskeleton and responsible for mediating cell–matrix adhesion³⁰. RGD sequences can bind to integrins through the β1 sub-unit³¹. Figure 7C showed that hWJ-MSC grown on spidroin micropattern expressed integrin β1. These results indicated that spidroin bioink was able to facilitate cell adhesion through interaction between RGD and integrin β1.

Micropattern width effect on glycosaminoglycan matrix deposition

Glycosaminoglycan (GAG) levels of hWJ-MSCs on fibronectin micropattern were quantified on day-7, 14, and 21 (Fig. 8). GAG is one of the main functional elements of cartilage that plays an important role in homeostasis. Not only does it provide mechanical resistance to pressure, but GAG is also involved in signaling pathways that regulate cell adhesion, proliferation, and differentiation³². The results showed that the GAG levels in the micropattern treatment with the size of 500 µm and 1000 µm increased significantly compared to the control group on day-21. Several in vitro MSCs chondrogenesis studies showed that GAG levels will tend to increase from day-14 to day-28^33,34,35.

A similar study on GAG content during MSCs differentiation with striped micropattern induction was done by Chou et al. (2013) using the same width size of 500 µm and 1000 µm, but the MSCs were cultured in chondrogenic medium condition³⁶. Chou et al. found that micropattern treatment cells had higher GAG levels compared to the control group, however, GAG content on 500 µm and 1000 µm groups was the same³⁶. This insignificant difference in GAG content can be caused by the usage of a chondrogenic medium that masked the effect of micropattern induction. Therefore, in this research, the cells were cultured in a basal medium without any chondrogenic supplementation. Our result showed that GAG levels did increase significantly on day-21 by as much as 56% for 500 µm and 72% for 1000 µm.

The role of spidroin micropattern from Argiope appensa to direct hWJ-MSC chondrogenesis has previously been investigated²². Hernando et al. showed that GAG level was highest in cells cultured on spidroin micropattern with 50 µg/ml spidroin concentration and 1000 µm micropattern width²². The spidroin concentration was the same as the fibronectin concentration used in this research (50 µg/ml). Our GAG result on fibronectin also proved that 1000 µm micropattern width could support chondrogenesis better than 500 µm size.

Upregulation of SOX9 expression in both mRNA and protein by spidroin micropattern

Spidroin micropattern treatment in both 500 µm and 1000 µm induced significantly high levels of SOX9 mRNA expression on day-21 (Fig. 9). On the other hand, fibronectin micropattern treatment has the same relative expression level as the control group. SOX9 expression is required for the initial step in mesenchymal condensation, differentiation, and proliferation of chondrocytes. Since SOX9 is a transcription factor that is essential and directly regulates collagen type II expression, SOX9 mRNA upregulation is thought to precede the expression of the COL2A1 gene³⁷. However, during chondrogenesis, SOX9 activity is necessary for several successive steps^37,38. The high relative expression of SOX9 mRNA in spidroin micropattern treatment during the late-stage of chondrogenesis (day-21) can be attributed to SOX9 function that inhibits proliferating chondrocytes transition into hypertrophy³⁸.

hWJ-MSCs grown on spidroin micropattern were able to maintain long-term induction of SOX9 gene expression compared to fibronectin. Studies showed that this long-term SOX9 activity could function to suppress RUNX2 expression, an osteogenic transcription factor, and therefore, prevents spidroin micropattern-induced cells from progressing into osteogenic fate^39,40. SOX9 mRNA upregulation marks the onset of chondrogenesis. Afterward, the SOX9 mRNA level started increasing from day-14 and reached its peak on day-21⁴¹. Looking at the immunocytochemistry result on SOX9 protein, micropattern treatment accelerates the synthesis of SOX9 protein which can already be detected from day-7 (Fig. 10). Figure 10 also showed the presence of SOX9 protein on day-14 and 21.

Spidroin micropattern induces signaling pathways that regulate SOX9 expression, which in turn will advance the differentiation step towards chondrocytes. Our data showed that the increased level of SOX9 gene expression in spidroin treatment is correlated with an increase in SOX9 protein synthesis, as confirmed with immunocytochemistry. The SOX9 protein was detected in both spidroin and fibronectin micropattern from day-7 and localized inside the nucleus. Subsequently, SOX9 acts as a master regulator that plays an important role in the transcription of pro-chondrogenic genes such as type II collagen (COL2A1 gene) and core protein of proteoglycan⁴².

Spidroin micropattern downregulates COL2A1 mRNA on late-stage chondrogenesis and induces protein expression

COL2A1 gene expression data on day-21 showed that the relative expression of the spidroin and fibronectin micropattern treatment was significantly lower compared to the control group (Fig. 11). Throughout chondrogenic differentiation, the expression of collagen type II mRNA occurs during day-7 until day-11⁴³. This explains why most of the COL2A1 mRNA in micropattern-induced cells on day-21 was decreased. The fact that the control group has higher COL2A1 relative mRNA expression on day-21, is an indicator of an incomplete or slow chondrogenic differentiation process⁴⁴. This is further verified by analyzing the protein expression of collagen type II using immunocytochemistry. Both the spidroin and fibronectin treatment groups started synthesizing collagen type II protein on day-14, in comparison to the control group in which the collagen type II protein was not detected even on day-21.

Based on the comparison of ICC results, it can be noted that SOX9 protein expression preceded collagen type II in micropattern treatment (Fig. 12). However, the relative expression of mRNA on day-21 did not show a correlation between SOX9 and COL2A1 gene expression. This may be due to SOX9 that regulates several steps of chondrogenic differentiation, and hence, a high level of SOX9 does not always increase COL2A1 mRNA nor collagen type II protein expression^45,46. Additionally, long-term expression of collagen type II does not require SOX9³⁷. Furthermore, the upregulation of the SOX9 gene in spidroin treatment occurred on late-stage chondrogenesis, in which collagen type II had already been synthesized.

Spidroin micropattern was able to promote chondrogenesis of hWJ-MSCs in vitro without chondrogenic inducers such as the TGF-β superfamily. Biomaterials containing RGD sequences can induce differentiation because the interaction between MSCs and RGD sequences mimics the cell microenvironment during chondrogenesis of limb bud development⁴⁷. Figure 13 showed the mechanism of spidroin micropattern on chondrogenesis. RGD sequences on spidroin interact with integrin β1 located on the cell membrane. Which then activates the expression of the SOX9 gene, the master regulator of pro-chondrogenic genes. Subsequently, SOX9 protein acts as a transcription factor and increases the expression of the COL2A1 gene that encodes collagen type II protein. Spidroin also increases GAG content which is a part of proteoglycan molecules that consists of core protein attached to the GAG chain.

Even though the exact signaling cascade from spidroin micropattern interaction with integrin β1 to the elevated expression of the SOX9 gene remains unclear, previous studies showed that integrin-linked kinase (ILK) and p38 MAPK might be involved. Perera et al. (2010) noted that integrin activation leads to phosphorylation of integrin-linked kinase (ILK), which in turn phosphorylate p38 MAPK⁴⁸. The MAPK signaling network is thought to control the response of micropattern induction on chondrogenic differentiation. Studies showed that p38 MAPK protein is responsible for regulating SOX9 expression level by maintaining the half-life stability of SOX9 mRNA^49,50.

Another factor that contributes to micropattern induction that resulted in chondrogenesis is the micropattern 1000 µm width. The micropattern size provides optimal cell-to-cell contact and promotes aggregation leading to chondrogenesis. Several studies proved that the membrane protein that facilitates interaction between cells and has a vital role in chondrogenic mesenchymal condensation is N-cadherin^51,52. According to previous studies, the formation of the N-cadherin complex with adjacent cells resulted in the phosphorylation of free β-catenin. Once phosphorylated, it becomes inactivated and will bind to the membrane adhesion complex. This β-catenin inactivation prevents β-catenin from inducing SOX9 degradation via proteasome that will inhibit chondrogenesis^53,54,55. Thus, based on other studies, micropattern width is thought to influence chondrogenic differentiation by a different signaling cascade from spidroin.

Overall, this research showed that spidroin can be used as a bioink for micropattern and induced chondrogenesis at the same level as fibronectin. Fibronectin has long been known as the ideal substrate for micropatterning due to the availability of RGD sequences in the protein. This study proved that Argiope appensa spidroin contains RGD sequences and enhances the hWJ-MSCs differentiation process as shown in the ICC and RT-qPCR results. Micropattern treatment restricts cells to a particular area, which leads to aggregation, a crucial step in chondrogenesis. Spidroin micropattern thus can be seen as a new approach for cartilage tissue engineering, to accelerate the chondrogenic differentiation of MSCs. Future study is required to elaborate more regarding the underlying molecular pathway that mediates the signals from integrin β1 to the expression of SOX9.

Thus far, micropatterning of cells is still limited to 2D surfaces. In this study, the micropatterning was used to differentiate hWJ-MSCs within a shorter period and determine the size of the pattern. Micropattern itself prevents further dedifferentiation of chondrocytes when cultured in 2D substrate⁵⁶. However, the structure of 3D native cartilage is different from micropatterned chondrocyte culture. Recent research showed that we can create micropattern surface in cell sheet technology and eventually stacking it to form complex 3D shapes⁵⁷. Multilayered cell sheets can be formed by magnetic technology such as through the attraction of MNP-labelled cells. However, this needs further research regarding the maintenance of this 3D structure, for example oxygen, nutrient, and also metabolite exchange.