Enhancement of osteogenic differentiation in umbilical cord mesenchymal stem cells using a silver nanoparticle-embedded carboxymethyl cellulose nano scaffoldAbstract
Introduction Some periodontal treatments seek to achieve complete and functional regeneration of periodontal tissues, but these interventions often have limited success. Hence, more predictable and effective alternatives are still being sought. The aim was to evaluate the potential for osteogenic differentiation and calcium deposition of umbilical cord stem cells on a carboxymethylcellulose (CMC) based nanofiber with silver nanoparticles (Ag-NPs) and polyvinyl alcohol (PVA).
Methodology Human umbilical cord mesenchymal cells (PCS-500-010) were cultured in the presence of a CMC nanofiber with Ag-NPs, and an analysis of calcium deposits with alizarin red staining and RT-qPCR for gene expression of the type I collagen, RUNX2, alkaline phosphatase, and osteocalcin was performed.
Results The findings showed that membrane 2 (M2) promoted the gene expression of alkaline phosphatase (R=2.214; P=0.0067), RUNX2 (R=2.041; P=0.004), and osteocalcin (R=1.315; P=0.039). Meanwhile, membrane 3 (M3) promoted the gene expression of type I collagen (R=2.169; P=0.042). The nanofibers induced the cells to form calcium deposits where the M3 showed the greatest amount (OD 450 nm=0.215).
Conclusions The CMC–AgNP/PVA nanofiber induced higher levels of gene expression and mineral deposition. These findings provide preliminary evidence that the nanofiber supports osteogenic differentiation–associated responses in vitro. However, further in vivo studies are required to confirm its efficacy.
Keywords
Cell differentiation; Nanofibers; Stem cells; Carboxymethylcellulose sodium; Nanoparticles
Introduction
Periodontitis is a chronic inflammatory disease of the periodontium associated with microorganisms and mediated by the host.1 Periodontitis affects over 1 billion people globally, ranking as a top-11 most prevalent disease (one of the most prevalent chronic conditions), generating considerable health costs.2 Its most severe forms affect approximately 11–19% of the population and, together with tooth decay and tooth loss, contribute to the $710 billion in global direct and indirect productivity costs.3,4
Periodontal disease is characterized by gingival inflammation, insertion loss, bone resorption, and formation of periodontal pockets.5,6 This is of great importance because loss of attachment caused by a bone defect affects the long-term prognosis of a dental organ. Such loss leads to tooth mobility, shifting, and increased bacterial accumulation, frequently resulting in tooth loss.7,8 Thus, the goal of periodontal treatment is to stop the progression of the disease and regenerate damaged structures to restore their function.9
Different treatments seek to achieve a complete and functional regeneration of periodontal tissues. However, these techniques have limited success. So, more predictable alternatives are still sought.9,10 Systematized studies conclude that, although certain combinations of regenerative materials favor clinical outcomes, an utterly predictable material that regenerates tissues is yet to be achieved.11
Cell therapy is a treatment that uses stem cells to replace or repair damaged tissues, including those in the periodontium. These cells can be differentiated into specialized cells with the necessary characteristics for each tissue type. They can be obtained from different sources, such as the umbilical cord, bone marrow, and dental pulp.15 It has also been shown that, compared to conventional periodontal regeneration therapy, the application of stem cells in tissue engineering presents a significant advantage in promoting and possibly completely regenerating tissues.16
On the other hand, tissue engineering is a multidisciplinary field with a promising focus on tissue restoration and reconstruction. Therefore, scaffolds are crucial in supporting cells and guiding their growth in a tissue for regeneration.17,18 This is why the development of nanofibers seems to have promising applications in dentistry by favoring cell adhesion, proliferation, and differentiation. Likewise, the high porosity of nanofiber scaffolds provides more structural space for the cells and facilitates the exchange of nutrients and waste between the scaffold and the environment.19
Different materials can be used to produce a nanofiber, and these are classified into natural and synthetic. The first is composed of collagen, gelatin, chitosan, chitin, cellulose, and starch. The latter are produced with lactic acid, glycolic, lactic-glycolic, caprolactone, and lactide-caprolactone.22 The derivatives of cellulose nanofibers include acetate-cellulose, methylcellulose, and carboxymethylcellulose (CMC). The latter has become the most important in manufacturing nanofibers due to its polyelectrolytic nature and biocompatibility.23
The use of CMC nanofibers with silver nanoparticles along with umbilical cord stem cells seems a promising treatment that has been previously evaluated for chronic wound disease. However, its potential to promote regeneration in bone defects is yet to be explored.24Previous research by Cienfuegos-Sarmiento, et al.25 (2023) proposed the use of nanofiber membranes based on polymer blends of a CMC/silver nanoparticle (CMC-AgNP) compound and polyvinyl alcohol (PVA) as suitable scaffolds for tissue engineering applications. They observed that the stiffness of the membranes increases with greater CMC-AgNP weight content, resulting in mechanical properties suitable for their application as tissue engineering scaffolds. They also observed low in vitro toxicity in Schwann cell cultures.
Because of the above, this study aimed to evaluate the osteogenic differentiation on umbilical cord-derived mesenchymal stem cells using a CMC–AgNP/PVA nanofiber developed and characterized by Cienfuegos-Sarmiento, et al.25(2023).
Methodology
Primary umbilical cord-derived mesenchymal stem cells were cultured in the presence of a CMC nanofiber with Ag-NPs for subsequent analysis of calcium deposits with alizarin red and qPCR regarding the expression of the COL1, RUNX2, alkaline phosphatase (ALP), osteocalcin, and β-actin genes.
Ethics statement
Primary umbilical cord-derived mesenchymal stem cells were purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA) (catalog no. PCS-500-010, lot no. 64310856). According to the provider’s documentation, umbilical cord tissue was collected under written informed consent from donors and with approval from an appropriate Research Ethics Committee/IRB at the collection site. Cells were supplied de-identified and accompanied by a certificate of analysis and quality/safety testing documentation. Because this study involved in vitro experiments using commercially obtained de-identified human cells and collected no new human tissue, no additional ethics approval was required.
Cell thawing and culturing
A volume of 1 mL of cell line (1×106 cells/mL) was thawed and cultured in basal mesenchymal cell medium with MSC supplement (2% fetal bovine serum, 5 ng/mL rh FGFb, 5 ng/mL rh FGFa, 5 ng/mL rh EGF) plus antibiotic-antifungal (100 units/mL penicillin G, 125 μg/mL streptomycin, 25 ng/mL amphotericin B).
For the initial stem cell culture, the cells were counted and placed in T25 cell culture vials up to a volume of 4 mL. Subsequently, the cell culture vials were incubated at 37ºC 5% CO2 for eight days up to the desired confluence (90%). After incubation, the cells were recovered using a conventional trypsinization protocol. Then, the cells were stained with 0.4% trypan blue and counted using a Neubauer chamber to obtain a concentration of 1×105 cells/mL.
Osteodifferentiation assay
For the osteodifferentiation assay, 100 µL of the cell suspension were seeded in each well, corresponding to 10,000 cells. This amount was placed in 50 of the 96 wells, according to Figure 1. The cells were cultured for 24 h at 37ºC. Then, 100 μL of mesenchymal basal medium (MBM) or StemMACS OsteoDiff (Miltenyi Biotec, GmbH, Bergisch Gladbach, Germany) were added as appropriate to achieve a working volume of 200 μL. The medium was distributed in the wells as follows (Figure 1): MBM (A1-A9 wells), MBM + CMC nanofibers (B1-B12 wells), MBM + TXT cytoplast nanofibers (C1-C4 wells), StemMACS OsteoDiff (D1-D9), StemMACS OsteoDiff + CMC membranes (E1-E12 wells), and StemMACS OsteoDiff + TXT cytoplast membrane (F1-F4 wells).
Design of the osteodifferentiation assay. The black circles represent the empty wells. The rest of the wells contained 100 μL of medium with PCS-500-010 mesenchymal cells. Moreover, 100 μL of basal medium (MBM) or StemMACS OsteoDiff were added up to a working volume of 200 μL with a cell concentration of 10,000 cells per well. MBM: mesenchymal basal medium. TXT: cytoplast TXT-200 membrane. M1-M3: CMC/PVA Ag Nanoparticles nanofibers.
The batch of membranes (M1, M2, and M3) developed and characterized by Cienfuegos-Sarmiento, et al.25 (2023) were used in this study. It had the following characteristics:
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Fiber diameter: M1= 312.0±76.0 nm, M2= 226.3±67.2 nm, and M3= 210.1±58.8 nm.
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CMC concentration: 53 mg/mL the same for all.
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AgNP concentration: M1= 2.8×10-4 AgNP/g, M2= 5.9×10-4 AgNP/g, and M3= 9.3×10-4 AgNP/g.
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PVA/CMC ratio (PVA:CMC-AgNP): M1= 9:1, M2= 8:2, and M3= 7:3
The manufacturing parameters for electrospinning were a 6-hour duration, 40-50% relative humidity, 10-kV voltage, 1-µL/min polymer mix flow rate, and a needle tip-to-collector distance of 8 cm.
The experiment was carried out in triplicate, which is why each of these nanofibers is three times in the same row. It was used to perform the PCR of the gene expression of COL1, RUNX2, ALP, osteocalcin, and β-actin. Similarly, a fourth well with each nanofiber was used to perform the alizarin red tests to detect calcium deposits. The culture medium was changed every four days, and the cultures were left at 37ºC for 21 days before the qPCR and alizarin red staining analysis.
Alizarin red staining
Wells B10 – B12, C4, E10 – E12, and F4 were intended to observe the presence of mineral deposits by alizarin red staining. On day 21 of incubation, the medium of each well was removed, and cells were washed with 1X PBS, and 10% formalin was placed to cover the cell monolayer. After 1 h, the cells were washed, and 100 μL of alizarin red was added to cover the cell monolayer and incubated at room temperature for 3 min. Then, the alizarin red stain was removed, and the cell monolayer was washed three times. The cells were observed under the microscope. Finally, the stained samples were treated with 10% acetic acid (100 μL of acetic acid for each well) and quantified in a 96-well microplate reader at 450 nm.
Gene expression assay and total RNA extraction
Wells A1-A3, B1-B9, C1-C3, E1-E9, and F1-F3 were used for the qPCR to assess the gene expression of COL1, RUNX2, ALP, osteocalcin, and β-actin. The incubator plate was taken for RNA extraction for the qPCR. Half was removed for each well and 50 μL of 1X PBS was placed in them. Subsequently, the 1X PBS was removed, and 50 μL of trypsin were added and left to incubate for 10 min at 37ºC. Then 150 μL of unsupplemented medium were added and transferred to plastic tubes to centrifuge at 300 × g for 10 min without acceleration or braking. Then, 1 mL of TRIzol was added; vortexed for 15 s, and left to stand for 15 min. Subsequently, 200 μL of chloroform were added; vortexed for 15 s, then left to stand for 15 min for subsequent centrifugation at 12,000 × g for 5 min. Once such time had passed, the aqueous phase containing the RNA was transferred to a 1.5 mL tube, and 500 μL of isopropanol were added and incubated for 10 min. The samples were centrifuged at 12,000 × g for 10 min; the supernatant was discarded, and the RNA precipitate was dried for 10 min. The RNA precipitate was suspended in 20 mL of nuclease-free water and incubated for 15 min at 60°C.
RNA reverse transcription
To synthesize the complementary DNA, the M-MLV Reverse Transcriptase kit (Promega, Madison, WI, USA) was used following the manufacturer’s instructions. First, four μL of RNA were obtained, 1 μL of random primer was added, and the mixture was incubated at 70ºC for 5 min. It was then cooled at 4ºC for another 5 min, and then the 1.5 mL tube was centrifuged at 12,000 × g for 5 min. For each sample, 4 μL of reaction buffer, 3 μL of MgCl2, 1 μL of DNTP mix, 1 μl of RT, and 6 μl of nuclease-free water were prepared. Then, 5 μL of the RNA + random primer mixture was added to reach a final volume of 20 μL. Finally, it was incubated at 25 °C for 5 min, at 42 °C for 1 h, at 70°C for 5 min, at 42°C for 1 h, and finally at 70°C for 15 min.
Gene expression
To analyze gene expression by real-time PCR, oligonucleotides targeting the genes of ALP, collagen type 1, Runx2, osteocalcin, and β-actin (as reference gene) were used. The primer sequences had been used for osteodifferentiation assays in previous studies with a >0.9 efficiency.26,27 CT values were obtained to evaluate the stability of the reference gene. The coefficient of variation was calculated for each experimental condition. Furthermore, the expression stability value (M) obtained from geNorm totaled 0.31, showing an acceptable reliability and stability for this study (Table 1).
The sequences are described as follows. Runx2 Fwd: 5’-CTTCATTTGCACTGGGTCAC-3’ (Tm: 58.4°C), Runx2 Rv: 5’-CTGCAAATCTCAGCCATGTT-3’ (Tm: 56.4°C); Collagen type I Fwd: 5’-ACCAACTGAACGTGACCAAA-3’ (Tm: 56.4°C), Collagen type I Rv: 5’-AGTGGGCAGAAAGGGACTTA-3’ (Tm: 58.4°C); osteocalcin Fwd: 5’-CTGCATTCTGCCTCTCTGAC-3’ (Tm: 60.5°C), osteocalcin Rv: 5’-CCGGAGTCTATTCACCACCT-3’ (Tm: 60.5°C); ALP Fwd: 5’-CGTCAATTAACGGCTGACAC-3’ (Tm: 58.4°C); ALP Rv: 5’-TCTGGCACAAATGAGTTGGT-3’ (Tm: 56.4°C); β-actin Fwd: 5’- GCTGTGCTATGTTGCCCTAGA-3’ (Tm: 64.0°C), and β-actin Rv: 5’-GGAACCGCTCATTGCCGATAG-3’ (Tm: 63.3°C).
The qPCR reactions were prepared with 2X qPCR Maxima Sybr Green master mix (Thermo-Scientific, Waltham, MA, USA), 200 nM of forward and reverse primer mix, 50 ng of cDNA, and nuclease-free water up to a final volume of 25 μL. All qPCR reactions were prepared on a 96-well plate.
The procedure was conducted using a LightCycler 480II thermal cycler (Roche, Basel, Switzerland), which was programmed with the Sybr Green I detection system into three stages: one pre-incubation cycle (95°C for 10 min), 40 amplification cycles (95 ° C for 10 s, 52°C for 15 s and 72°C for 10 s in individual acquisition mode), one melting curve cycle (95 ° C for 5 s, 65 ° C for 1 min, and 97°C in continuous acquisition mode), and one cooling cycle (4°C for 30 s). For relative expression analysis, threshold cycle values (Ct, cycle threshold) and normalized ratios (R) were calculated by the ΔΔCt method on LightCycler 480II (Roche, Basel, Switzerland) and Rest2009 (v2.0.13, QIAGEN, Hilden, Germany). Relative expression is based on the expression ratio of a target gene to a reference gene (β-actin). The gene expression values of the treatments were compared with cells without treatment and treated with StemMACS OsteoDiff as a negative and positive control of osteodifferentiation, respectively.
Gene expression experiments using RT-qPCR were performed in technical triplicate within a 96-well plate in three independent biological experiments (Figure 1). Alizarin red staining was performed in only three independent biological experiments. Cells were obtained from a commercial batch provided by the ATCC and cultured to the P2 passage to preserve their multilineage potential and prevent senescence. The wells were predefined in each experiment to maintain a consistent experimental design. Furthermore, OD measurements derived from alizarin red staining were performed blinded by three independent analysts in each biological experiment
Statistical analysis
In the alizarin red assay data, the Shapiro-Wilk and Levene’s tests were performed to determine data distribution and variance homogeneity, respectively. Then, to establish differences between the experimental conditions, a one-way analysis of variance and a post hoc Tukey’s honest significant difference test were performed for multiple comparisons. Moreover, gene expression (R) differences were assessed using the relative expression software tool (REST 2009) with a statistical approach based on the Pfaffl method combined with a randomization test for statistical significance (P).28 A p-value <0.05 was considered statistically significant in all treatments. R, standard error, and 95% CI were calculated. The SPSS statistical package (v.22, IBM, North Castle, NY, USA) and MS Excel (2017, Microsoft, Redmond, WA, US) were also used.
Results
Gene expression
The expression of osteogenic markers such as collagen, osteocalcin, RUNX2, ALP, and calcium deposits were analyzed 21 days after the initial culture.
The gene expression (R) of collagen in the presence of the CMC nanofibers M1, M2, and M3 showed R = 1.157, R = 1.001, and R = 2.169, respectively, whereas the expression in the presence of the TXT membrane, R = 0.92; in the presence of only StemMACS OsteoDiff, R=1.871; and in the presence of StemMACS OsteoDiff with the nanofiber, R = 1.269, R = 2.331, and R = 2.417 for M1, M2, and M3, respectively. Finally, the StemMACS OsteoDiff in the presence of the TXT membrane (StemMACS OsteoDiff + TXT) showed R = 1.114. In the analysis with the p-value, a significant difference was found in the expression of the collagen gene in M3, StemMACS OsteoDiff, StemMACS OsteoDiff + M1, StemMACS OsteoDiff + M2, StemMACS OsteoDiff + M3, which overexpressed it. Thus, it is possible to determine that the presence of the nanofiber M3 increases the gene expression of collagen and, although the StemMACS OsteoDiff alone also enables a greater gene expression, this is potentiated when this means of differentiation is combined with the nanofiber M3, obtaining a synergism (R = 2.417), as shown in Table 2.
The gene expression of osteocalcin in the presence of CMC nanofibers M1, M2, and M3 showed R = 1.083, R = 1.315, and R = 0.871, respectively; whereas the expression in the presence of the TXT membrane, R = 1.016; in the presence of StemMACS OsteoDiff, R=1.94; and in the combination of StemMACS OsteoDiff with nanofiber M1, R=1.555; M2 totaled R=3.555 and M3 equaled R=1.655. Finally, the StemMACS OsteoDiff with TXT membrane showed R=1.64. Analyzing these results showed a significant difference in the overexpression of the osteocalcin gene in the presence of M2, StemMACS OsteoDiff, StemMACS OsteoDiff + M1, StemMACS OsteoDiff + M2, StemMACS OsteoDiff + M3, and in StemMACS OsteoDiff + TXT. Thus, it is possible to determine that the nanofiber M2 increases the gene expression of osteocalcin (R = 1.315) and, although the StemMACS OsteoDiff alone also increased gene expression (R = 1.94), this is potentiated when this means of differentiation is combined with nanofiber M2, obtaining a synergism (R = 3.555), as shown in Table 3.
The gene expression of RUNX2 in nanofiber M1 totaled R = 0.199; that of M2, R= 2.041; and that of M3, R = 0.028. On the other hand, gene expression in the presence of the TXT membrane alone obtained R = 0.873. In the presence of StemMACS OsteoDiff, R = 1.877, whereas in the combination of StemMACS OsteoDiff with nanofiber M1, R = 1.299; M2, R = 3.255; and M3, R = 1.231. Finally, StemMACS OsteoDiff with the TXT membrane obtained R = 1.867. Based on the p-value, it was determined that there was a significant overexpression of the RUNX2 gene in the presence of nanofiber M2, StemMACS OsteoDiff alone, StemMACS OsteoDiff with nanofiber M1, StemMACS OsteoDiff with M2, StemMACS OsteoDiff with nanofiber M3, and StemMACS OsteoDiff with TXT membrane. On the other hand, nanofiber M1 and M3 showed a decrease in the expression of this gene. Although the presence of the nanofiber M2 and StemMACS OsteoDiff alone increased the gene expression of RUNX2 (R = 2.041 and R = 1.877, respectively), their use in tandem more greatly expressed that gene, creating a synergism (R = 3.255) (Table 4).
The gene expression of ALP in the presence of CMC nanofiber M1, M2, and M3 totaled R = 1.083, R = 2.214, and R = 0.895, respectively, whereas in the presence of the TXT membrane, R = 1.079; in the presence of StemMACS OsteoDiff, R=1.984; in the combination of StemMACS OsteoDiff with nanofiber M1, R=1.498; with M2, R=3.447; with M3, R=1.598; and with the StemMACS OsteoDiff and the TXT membrane, R=1.968. In the analysis with the p-value a significant difference was found in the expression of the ALP gene in M2, StemMACS OsteoDiff, StemMACS OsteoDiff + M1, StemMACS OsteoDiff + M2, StemMACS OsteoDiff + M3, and StemMACS OsteoDiff + TXT, which overexpressed this gene. The presence of nanofiber M2 increased the gene expression of ALP (R = 2.214) and, although StemMACS OsteoDiff alone also increased gene expression (R = 1.984), this is potentiated when this means of differentiation is combined with the nanofiber, especially with nanofiber M2, obtaining a synergism (R = 3.447) (Table 5).
Likewise, it is essential to note that the presence of the TXT membrane alone failed to increase the gene expression of any of the four genes in this study, showing the potential for osteodifferentiation of the CMC–AgNP/PVA nanofiber.
Calcium deposits
The 450-nm optical density of calcium deposits using alizarin red is shown in Figure 2.
The optical density values at 450 nm from alizarin red assays. M: nanofiber. The data represent the mean and standard deviation of three replicates of independent experiments. Additionally, statistically significant differences (P<0.05) are indicated with an asterisk. TXT: cytoplast TXT-200 membrane. MBM: mesenchymal basal medium.
The results were statistically compared by one-way analysis of variance with Tukey’s honest significant difference. The production of calcium deposits was observed in the basal mesenchymal medium (OD 450 nm = 0.011± 0.005). A statistically significant higher value (p<0.05) was observed when using the MBM with nanofibers M1, M2, and M3 (OD 450 nm = 0.131± 0.005, OD 450 nm = 0.126 ± 0.0048 and OD 450 nm = 0.192 ± 0.0042, respectively). Similarly, a statistically significant difference (P<0.05) was found when using one of the three nanofibers in the presence of StemMACS OsteoDiff (M1: OD 450 nm = 0.155 ± 0.0036; M2: OD 450 nm = 0.16 ± 0.0039; M3 OD 450 nm = 0.215 ± 0.0037) compared to the use of these, but in MBM. Likewise, a statistically significant greater difference was found in the use of nanofibers in MBM and StemMACS OsteoDiff when compared to the presence of the TXT membrane in either of these media (MBM +TXT: OD 450 nm = 0.064 ± 0.0037 and StemMACS OsteoDiff + TXT: OD 450 nm = 0.021 ± 0.0046).
Like gene expression, the results show that using the TXT membrane seems to scarcely affect calcium deposits as CMC nanofibers.
Discussion
CMC is a hydrophilic polysaccharide derived by chemically modifying cellulose with chloroacetate in an alkaline environment. This modification introduces carboxymethyl groups at the C2, C3, or C6 positions of glucose units.29 The negatively charged, water-soluble nature of CMC enables it to chelate multivalent cations, such as Ca2+. Polymeric nanofibers have been developed to increase the bioactivity of polymeric nanomaterials. Singh, et al.30 (2016) created CMC plus silk fibroin composite nanofibers to enhance biomineralization in silk fibroin bone scaffolds. Electrospun into nanofibers, these materials improve osteointegration by closely mimicking the natural extracellular matrix. CMC aids the uniform nucleation and formation of spherical calcium phosphate crystals on the scaffold via its COOH and OH functional groups, facilitating calcium cation binding. Although CMC significantly improved biomineralization and water uptake in the CMC plus silk fibroin scaffold, it failed to affect its mechanical properties. Moreover, culturing mesenchymal stem cells on this biomimetic CMC plus silk fibroin substrate differentiated them into the osteogenic lineage. This study observed that the nanofiber can support the development of a scaffold that enabled osteogenic differentiation and that the thickness can influence these effects.
Martínez-Rodríguez, et al.31 (2016) developed a CMC nanofiber with silver nanoparticles and PVA by electrospinning aqueous solution mixtures of compound CMC-silver nanoparticles and PVA, which were prepared in different proportions by weight of CMC/ PVA. The authors mention that this is the thinnest nanofiber in the literature and that it has a potential application in manufacturing antibacterial scaffolds for tissue engineering, which can be applied in areas such as medicine and dentistry. This nanofiber was used in this study, making cellular biocompatible by enabling the osteodifferentiation of stem cells and their culture for 21 days, confirming its potential use in regenerative materials in periodontics.
Similarly, Laredo-Naranjo, et al.32 (2016) determined the antibacterial effect of silver nanoparticles in a CMC matrix on titanium plates at 16, 50, and 100% concentrations. To these cultures Streptococcus mutans and Porphyromonas gingivalis were added; 0.12% chlorhexidine was used as a control. The authors found that silver nanoparticles in all concentrations were antimicrobial, with concentrations being 50 and 100% more cytotoxic with 4% cell viability. On the other hand, 16% of silver nanoparticles had a 95% cell viability, less cytotoxic than 0.12% chlorhexidine. This study could suggest that silver nanoparticles in a CMC nano scaffold are helpful as an antibacterial scaffold by enabling the osteodifferentiation of umbilical cord stem cells without morphogenic changes. However, evaluating cytotoxic activity requires future research.
Vimalraj, et al.33 (2016) synthesized a scaffold containing CMC, nanohydroxyapatite, and ascorbic acid for bone tissue engineering applications. The ability for the cellular and molecular osteodifferentiation of the scaffold and a bioactive molecule (microRNA-15b, which acts as a post-transcriptional gene regulator and mediates osteoblast differentiation) was determined using mouse mesenchymal stem cells. The authors found that this scaffold promoted osteoblast differentiation, which they verified by an increase in calcium deposits and the expression of Runx2, ALP, and type I collagen in cultures with only half Dulbecco’s modified eagle medium and the nano scaffold without the micro-RNA. These results are comparable with this research regarding the capability of cell differentiation in the presence of CMC, with the subsequent mineralization and increase in the expression of osteodifferentiation genes, this component being biocompatible and with potential choice for use in regenerative materials.
Likewise, Teti, et al.34 (2015) published a study on the osteogenic and odontogenic differentiation of dental pulp stem cells grown in a hybrid CMC-hydroxyapatite hydrogel. This was done by culturing stem cells in a hybrid CMC-hydroxyapatite hydrogel and CMC hydrogel for one, three, five, seven, 14, and 21 days, performing cell viability tests and ultra morphological analyses to evaluate biocompatibility and cell adhesion. Using real-time PCR, the authors found increased gene expression of RUNX2 and type 1 collagen. Similarly, our results show an increase in gene expression of both genes in the presence of a CMC nanofiber when compared to the control membrane (TXT). So, it can be considered a promising candidate for periodontal tissue engineering.
Gorgieva, Girandon, and Kokol35 (2017) developed gelatin scaffolds enriched with cellulose nanofibrils to evaluate the growth of mesenchymal stem cells. The authors observed that all scaffolds containing this nanofibril resulted in more extensive Ca2+ deposition than the positive control or pure gel scaffold. Like them, the results in this research show that CMC nanofibers more significantly affect calcium deposits than the control group, in this case, the TXT membrane.
Qi, et al.36 (2018) fabricated non-woven sheets of CMC loaded with calcium phosphate (CaP) and investigated its potential to induce in vitro and in vivo differentiation of human mesenchymal cells into osteoblasts in mice. The authors found that in the absence of CaP, human mesenchymal cells in the CMC lamina had only enhanced ALP expression, indicating early osteoblast differentiation. Likewise, in vivo, they found new bone with cells expressing the transcription factor Sp7/Osterix, essential for osteoblast differentiation. As in this study, the potential of CMC membranes to increase the gene expression of osteogenic markers is demonstrated, albeit preliminarily. However, because this study is limited to in vitro assays, it shows no bone/periodontal regeneration, scaffold integration, vascularization, immune response, or functional tissue formation.
Using a dog lateral femoral condyle defect model, Sakamoto, et al.37 (2019) studied the bone regeneration capabilities of a nonwoven sheet of calcium phosphate (CaP)-loaded CMC (CMC/CaP). The authors found that the rate of new bone formation in the CMC/CaP group was significantly higher than in the control and CMC groups based on microcomputed tomography and histological evaluation. Therefore, it was concluded that these sheets promote new bone formation and are helpful as a substitute for bone grafting. In this case, our in vitro experiment supports the use of CMC for osteogenic differentiation. Therefore, it would be essential to continue this study and see whether the CMC nanofiber in the presence of silver nanoparticles and PVA would be effective for in vivo osteodifferentiation.
Previous studies have indicated that combining CMC+PVA+AgNPs incorporated into nanofibers could provide satisfactory results as a scaffold in tissue engineering.38 Consequently, in our laboratory, Cienfuegos-Sarmiento, et al.25 (2023) developed and characterized this combination of nanofibers, which showed low toxicity and microbial activity on Schwann cells. In this study, despite no morphological changes in the cells, it will be relevant to evaluate its effect by cytotoxicity assays in future research. The presence of silver nanoparticles warrants careful evaluation of dose- and time-dependent effects. While this study focused on osteogenic differentiation outcomes, comprehensive biocompatibility assessment (cell viability/proliferation, inflammatory signaling, oxidative stress, and long-term cellular compatibility) was not performed and should be addressed in future studies. Importantly, these biological endpoints should be correlated with quantitative Ag release measurements under the same culture conditions to establish exposure-response relationships and safety margins.
Recent studies, such as Lee, et al.39 (2024) have shown that CMC can act as an excellent scaffold when combined with other bioactive compounds such as fibroin, promoting osteodifferentiation. In that study, ALP activity in mesenchymal stem cells was significantly higher in the CMC scaffold, showing increased expression of osteocyte marker genes and proteins than the silk scaffold alone. These results are consistent with the ones in this study: increased ALP expression in CMC nanofiber M2.
Although the scaffold was produced using the same formulation and batch as in our previous characterization study, key parameters influencing biological performance (such as silver ion release kinetics under culture conditions, degradation behavior, mechanical stability during incubation, surface physicochemical properties, and long-term scaffold–cell interactions) were not directly evaluated in this work. These factors may modulate osteogenic differentiation and represent important limitations. Future work will quantify Ag release, degradation and pH changes, mechanical properties after incubation, and surface properties before/after culture.
Conclusion
The osteogenic differentiation of umbilical cord-derived mesenchymal stem cells was successfully stimulated in the presence of the CMC–AgNP/PVA nanofiber and in the control group using the TXT membrane. Furthermore, the CMC–AgNP/PVA nanofiber demonstrated the ability to promote the expression of key osteogenic markers (including type I collagen, ALP, osteocalcin, and RUNX2) alongside the stimulation of calcium deposition, thereby supporting the proposed research hypothesis. Notably, the CMC–AgNP/PVA nanofiber induced higher levels of gene expression and mineral deposition than the TXT membrane. Nevertheless, because this study is limited to in vitro assays, it showed no bone/periodontal regeneration. Therefore, the regenerative potential of the CMC–AgNP/PVA nanofiber remains preliminary. Confirmation requires appropriate in vivo models.
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