Biomaterial Stiffness of Wharton’s Jelly-Derived Mesenchymal Stem Cell-Conditioned Medium Modulates Fibroblasts Proliferation and Migration: A Preliminary Study

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Mesenchymal stem cells (MSCs) secrete a wide range of growth factors, and cytokines in response to environmental cues, thereby providing a microenvironment that promotes fibroblast migration and proliferation during wound healing. Recent studies have revealed that the mechanical microenvironment, especially stiffness, affects MSCs by determining their secretome, differentiation, or senescence¹,²,³. However, only few studies have investigated whether substrate stiffness modulates the wound healing potential of MSC-conditioned media²,³. In this preliminary study, we examined the therapeutic potential of chemically defined conditioned media (CDCM) produced from human umbilical cord Wharton’s jelly derived MSCs (wjMSCs) to promote wound healing in vitro. We compared CDCM derived from wjMSCs cultured on soft (500K Pascal [Pa]) or stiff (3G Pa) substrate.

wjMSCs (Kang Stem Biotech, Seoul, Korea) were cultured in xenofree HUMA II media (ROKIT Healthcare, Seoul, Korea) supplemented with human platelet lysate (HPL) (PL Bioscience, Aachen, Germany), 1 × Penstrep (GIBCO, Carlsbad, CA, USA), and 1 × GlutaMAX™ (GIBCO), and incubated in a 5% CO₂ incubator (Thermofisher, Waltham, MA, USA) at 37°C. HUMA II basal medium was used for CDCM. Passage 5 wjMSCs were seeded at 50,000 cells/cm² and cultured in HUMA II complete media until they reached 80% confluency. wjMSCs were seeded and cultured on two substrate types: a 500 kPa stiffness substrate and a 3 GPa tissue culture plastic surface. The culture media were then switched to either HUMA II basal medium or low-glucose Dulbecco’s modified Eagle’s medium (DMEM; GIBCO), serving as a negative control. After 24 hours, the culture medium was removed and replenished. Conditioned media were collected every 3 days during the 9 days of culture. Each conditioned medium was filtered with 0.45 um and 0.2 um filter (Thermofisher) sequentially and stored at 4°C until further test and analysis. Series dilutions of 500 kPa-CDCM were made for the proliferation test.

Human neonatal dermal fibroblasts (NDFBs) (Pelobiotech, Planegg, Germany) at passage 5 were cultured in xenofree HUMA II media supplemented with HPL, 1 × Penstrep, and 1 × GlutaMAX™, and incubated in a 5% CO₂ incubator at 37°C and 95% humidity. On 6-well plates, 2.5 × 10⁵ cells/ml or 2600 cells/cm² were seeded with complete HPL supplemented NDFB media and incubated at 37°C overnight. DMEM served as a negative control, and complete NDFB medium, DMEM supplemented with 1 × GlutaMAX™, and 2% fetal bovine serum (FBS) served as a positive control.

To investigate the effect of CDCM on NDFB proliferation, 500 kPa-CDCM and 3 GPa-CDCM were diluted in HUMA II basal medium under various conditions (100%, 50%, 30%, and 10%). Each well was washed three times with 1 × PBS to remove the complete NDFB media, and 3 ml of the assigned media for each group was added (n=5). The proliferation assay was performed for 7 days, and the medium was changed every 3 days. After 7 days, the cells were fixed with 4% paraformaldehyde and stained with diamidino phenylidole (DAPI) for cell counting. Image J software (NIH, Bethesda, MD, USA) was used for counting DAPI-stained nuclei.

For in vitro wound healing assay, NDFBs at passage 5 were cultured in xenofree HUMA II media supplemented with fibrin depleted HPL, 1 × Penstrep, and 1 × GlutaMAX™, and incubated in a 5% CO₂ incubator at 37°C and 95% humidity. In each 6-well plate, a sterile polydimethylsiloxane rubber block (20×6×4 mm) was firmly attached to the bottom of each well. NDFBs were seeded at 50,000 cells/cm² and incubated until they reached full confluency. Rubber blocks were removed without disturbing the cell alignment, and a 20 mm × 6 mm rectangular wound area was created for cell migration or in vitro wound closure. Next, 3 ml of conditioned medium was added. Migration was measured through the migration measurement window from day 1 (24 hours post-seeding) and then every 48 hours until day 11. The distance between the linear cell walls was measured using a microscope (Nikon, Tokyo, Japan), and the images were reconstituted using Image J software. All data were analyzed using the SPSS Statistics software (version 20.0; IBM Corp., Armonk, NY, USA). The Mann-Whitney U test was used to determine statistical differences in the values between the two groups. A p-value of less than 0.05 was set to indicate statistical significance.

Fig. 1 shows the results of the proliferation assays of NDFBs cultured in 500 kPa- or 3 GPa-CDCM diluted to concentrations of 100%, 50%, 30%, and 10%. NDFBs cultured in 100% 500 kPa-CDCM showed a significantly higher total cell count than those cultured in FBS-supplemented complete NDFB medium (positive control) or DMEM (p<0.05). The proliferation of NDFBs dramatically reduced when grown with diluted concentrations of 500 kPa-CDCM (50%, 30%, and 10%). NDFBs cultured with 100% 500 kPa-CDCM showed a higher total cell count than those cultured with 100% 3 GPa-CDCM (p<0.05)

Fig. 1. Human NDFBs proliferation assay. (A) NDFBs cultured with 500 kPa- or 3 GPa-CDCM diluted into the concentrations of 100%, 50%, 30%, and 10%. After 7 days of culture, the proliferation of NDFBs were promoted by 500 kPa-chemically defined conditioned media (CDCM) in a dose-dependent manner. (B) The number of NDFBs in microscopic ROI after 7 days of culture. NDFBs cultured with 100% 500 kPa-CDCM showed a significantly higher number of cell count compared with NDFBs cultured with 100% 3 GPa-CDCM, conventional complete NDFB media or DMEM CM (n=5).

NDFB: neonatal fibroblast, CDCM: chemically defined conditioned media, DMEM: Dulbecco’s Modified Eagle Medium, CM: conditioned media, ROI: region of interest.

^*p<0.05; Scale bar=500 μm.

Fig. 2 shows the results of in vitro wound healing assay. In the presence of CDCM, NDFBs covered the wounds more rapidly than in the control group. From day 5, NDFBs treated with either 500 kPa-CDCM or 3 GPa-CDCM had a shorter distance between the linear cell walls than that of NDFBs treated with DMEM (p<0.05). We further found a significant increase in the migration rate of NDFBs treated with 500 kPa-CDCM compared to those treated with 3 GPa-CDCM (p<0.05).

Fig. 2. In vitro wound healing assay. (A) Schematic description on the procedure. After human NDFBs were seeded and incubated until full confluency, rubber blocks were carefully removed not to disturb cell alignment and created 20×6 mm rectangular wound area for cell migration. (B) Microscopic photographs showing the migration measurement window. Photographs of the region behind the dotted line were not taken as they were not counted for migration distance measurement. (C) The distance between the linear cell wall from day 1 to 11. From day 5, the distance was shorter in the NDFBs treated with either 500 kPa- CDCM or 3 GPa-CDCM than that of NDFBs treated with DMEM CM. From day 7, a significant increase in the migration rate was observed in the NDFBs treated with 500 kPa-CDCM compared with NDFBs treated with 3 GPa-CDCM (n=3).

NDFB: neonatal fibroblast, CDCM: chemically defined conditioned media, DMEM: Dulbecco’s Modified Eagle Medium, CM: conditioned media.

The biophysical microenvironment influences MSCs to determine their secretome, differentiation, or senescence¹,²,³. MSCs sense the stiffness of extracelluar matrix via α and β integrin receptors by binding with its ligands⁴. The integrin-mediated mechanotransducers form the stress fibers by actin polymerization and elongation, consequently regulating the fate of MSCs⁵.

In this preliminary study, we found that CDCM derived from wjMSCs cultured on soft (500 kPa) substrate promote the proliferation and migration of NDFBs better than stiff (3G Pa) substrate. Our results indicate that environmental cues during the production of CDCM can affect its regenerative capacity. Further research is required to investigate the optimal mechanical and biochemical conditions for MSC cultures to produce a secretome profile that efficiently accelerates wound healing.