Stimulated Human Umbilical Cord Mesenchymal Stem Cells Enhance the Osteogenesis and Cranial Bone Regeneration through IL-32 Mediated P38 Signaling Pathway

Objective Our previous study found that it could significantly increase the expression of IL32 after stimulating the human umbilical cord mesenchymal stem cells (S-HuMSCs). However, its role on the osteogenesis and cranial bone regeneration is still largely unknown. Here, we investigated the possible mechanism of this effect. Material and Methods. A series of experiments, including single-cell sequencing, flow cytometry, quantitative real-time polymerase chain reaction, and western blotting, were carried out to evaluate the characteristic and adipogenic–osteogenic differentiation potential of IL-32 overexpression HuMSCs (IL-32highHuMSCs) through mediating the P38 signaling pathway. Moreover, a rat skull bone defect model was established and treated by directly injecting the IL-32highHuMSCs to conduct its role on the cranial bone regeneration. Results In total, it found that compared to HuMSCs, IL32 was significantly increased and promoted the osteogenic differentiation (lower expressions of PPARγ, Adiponectin, and C/EBPα, and increased expressions of RUNX2, ALP, BMP2, OPN, SP7, OCN, and DLX5) in the S-HuMSCs (P < 0.05). Meanwhile, the enhanced osteogenic differentiation of HuMSCs was recovered by IL-32 overexpression (IL-32highHuMSCs) through activating the P38 signaling pathway, like as the S-HuMSCs (P < 0.05). However, the osteogenic differentiation potential of IL-32highHuMSCs was significantly reversed by the P38 signaling pathway inhibitor SB203580 (P < 0.05). Additionally, the HuMSCs, S-HuMSCs, and IL-32highHuMSCs all presented adipogenic–osteogenic differentiation potential, with higher levels of CD73, CD90, and CD105, and lower CD14, CD34, and CD45 (P > 0.05). Furthermore, these findings were confirmed by the rat skull bone defect model, in which the cranial bone regeneration was more pronounced in the IL-32highHuMSCs treated group compared to those in the HuMSCs group, with higher expressions of RUNX2, ALP, BMP2, and DLX5 (P < 0.05). Conclusion We have confirmed that S-HuMSCs can enhance the osteogenesis and cranial bone regeneration through promoting IL-32-mediated P38 signaling pathway, which is proved that IL-32 may be a therapeutic target, or a biomarker for the treatment of cranial bone injuries.


Introduction
With the rapid development of economy and population aging, cranial bone injury has become a serious health issue with an ever-increasing prevalence, as it has estimated that in China, its occurrence has been nearly 100-200 per hundreds of thousands of people [1,2].Recently, the mainly existing treatments of cranial bone injury are included the autologous and allogeneic bone transplantation [3].Otherwise, there are many disadvantages of these clinical usages for the limited sources, invasive, immune rejection, and insufficient osteogenic ability [4].So in this condition, HuMSCs, as the common pluripotent stem cells, have become a seed candidate in the regenerative clinical medicine because of their easy separability, high moving trends, multidirectional differentiation potential, low immunogenicity, and noncytotoxicity [5].Commonly, HuMSCs can differentiate into different precursor cells under the relevant conditioned media, in which it is balanced of adipogenic-osteogenic differentiation programs.Nowadays, a large number of clinical studies have shown that local transplantation of HuMSCs has a good improvement effect on the bone defects and osteoporosis through increasing the expressions of TGF-β1, Runx2, osteoblasts, Wnt10b, and so on [6,7].Moreover, the animal experiments also have proved that tail vein injection of HuMSCs can promote the reconstruction of cranial bone injury through increasing the alkaline phosphatase (ALP) activity and reduce the contents of triglyceride [8,9].Otherwise, there are still some studies that were obtained the different consequences, which point out that HuMSCs can not form the ectopic new bone in the subcutaneous tissues under the natural state [10,11].Therefore, it is very urgent and important to discuss the methods for improving the osteogenetic ability by the related postprocessing.
As we all known, the balances of adipogenic-osteogenic differentiation of HuMSCs can be affected by the microenvironment [1,5].Meanwhile, some scholars had found that pretreatment of HuMSCs with proinflammatory cytokines, as the stimulated HuMSCs (S-HuMSCs), could enhance their immunosuppressive properties [10].However, no reports were discussed for the roles of S-HuMSCs on the osteogenesis and cranial bone injury in vivo and in vitro experiments.As demonstrated by our previous single-cell sequencing data, compared with the HuMSCs, S-HuMSCs lead to many aberrant expressions of related genes to enhance their biological functions and immune regulatory ability, in which there was a significant increase in the expression of IL-32 (HuMSCs) in the S-HuMSCs.Here, we found that IL-32, known as NK4, were contained in "RGD" motif-containing attachment sequence and it was a member of cytokines to induce the proinflammatory cytokines such as IL-1β, TNF-α, IL-6, and IL-8 [12,13].Additionally, a number of studies have revealed that actual participation of IL-32 depends on a series of downstream signaling activation events, as PI3K-AKT, NF-κB, P38mitogen-activated protein kinases (MAPK), ERK-MAPK, JNK-MAPK, and STAT1 pathways [14].Moreover, it has also shown that when IL-32 was added to PBMC cultures, it could maintain with soluble receptor activator of RANKL.Although the numbers of newly generated osteoclasts were increased, the percentage of lacunar resorption was significantly decreased, suggesting a possible inhibitory effect of IL-32 on the osteoclast activation [15].Meanwhile, some researches also have revealed the participation of IL-32 in the immune diseases, inflammatory reactions, and angiogenesis [16,17].However, IL-32, as an important regulatory factor in the S-HuMSCs, is involved in the osteogenesis and cranial bone injury, particularly the aggressiveness mediated by the immune microenvironment through the downstream signaling pathway, which is largely unknown.Therefore, this study aimed to verify the hypothesis that S-HuMSCs could enhance the osteogenesis and cranial bone regeneration by upregulating the IL-32/P38 signaling pathway.

Single-Cell
Sequencing of HuMSCs and S-HuMSCs.The samples of HuMSCs and S-HuMSCs (n = 3/group) were prepared in the cell suspensions and subjected to a series of multiple dilutions to make the cells under a single cell state.Then the whole mRNA was extracted and reverse transcribed into cDNA by the reverse transcriptase, and the gene expressions and immune repertoire data were obtained sequence with green fluorescent protein (GFP) and puromycin resistance gene, while the control vectors (NC) were the noload plasmid with only the GFP and puromycin resistance gene.These were also constructed and harvested by the Genepharma (Shanghai, China).Exactly, the HuMSCs were seeded in the six-well plate at 1 × 10 5 cells/well.Then they were respectively transferred by the IL-32 lentiviral and no-load plasmid vectors to respectively obtain the IL-32 high HuMSCs and NC-HuMSCs (1 × 10 7 TU/mL, MOI = 10) when the HuMSCs were grew to 60%, with the no lentivirus HuMSCs as the controls (HuMSCs).After 24 hr of transfer-ration, IL-32 high HuMSCs and NC-HuMSCs were replaced with a complete α-MEM medium containing puromycin (2 mmol/L) for 48 hr, which then were changed into α-MEM medium without puromycin when the degrees of HuMSCs were reached to 90%.The transfection efficiency of IL-32 high HuMSCs and NC-HuMSCs, as evaluating the contents of GFP were observed by the fluorescence microscope and flow cytometry.Furthermore, the transfection efficiency of target genes (IL-32) was detected by the quantitative real-time polymerase chain reaction (RT-PCR) and western blotting (WB).

Colony Forming Unit (CFU) and Proliferation of IL-32 high
HuMSCs.The HuMSC, NC-HuMSCs, and IL-32 high- HuMSCs were seeded in the six-well plate as 500 cells/well, and the complete α-MEM medium was changed every 3 days.After 10 days treatment, the medium was discarded and washed twice with phosphate buffered saline (PBS), which were then added and fixed with 1% paraformaldehyde for 15 min.They were afterwards rinsed twice with PBS and 1 mL crystal violet staining solution (Beyotime, China) was stained for 30 min.
Then the numbers of colonies were counted by the Image J software.Moreover, the HuMSC, NC-HuMSCs, and IL-32 high- HuMSCs were also seeded in the 96-well plate as 2,000 cells/well with three duplicates, then 10 μL CCK-8 (Dojindo, Japan) detection solution was added and detected by the microplate reader after the treatment for 9 days.
2.6.Animal Model of Cranial Bone Defect.Five 8-week-old Wistar rats were anesthetized by injecting 1% pentobarbital (0.6 mL/ 100 g), and two cranial bone lacunae were obtained using the dental drill (diameter: 5 mm) on two sides of midcranial suture.Then 5 × 10 5 NC-HuMSCs (left lacunae) and IL-32 high HuMSCs (right lacunae) were treated with the above cranial bone lacunae.At the 8 th week after the operation, the rats were sacrificed, then the skull was removed, fixed, and observed by the Micro-CT to observe the area of new bone formation.Meanwhile, the newly formed bone samples were taken to measure the expressions of genes on the osteogenic differentiation.

2.7.
Effects of IL-32 on the MAPK Signaling Pathways.The protein expressions of MAPK signaling pathways such as P38, ERK and JNK,in the HuMSCs, NC-HuMSCs, and IL-32 high HuMSCs were detected by WB method.Then the interactions between IL32 and P38 were measured by the CO-IP method [18].Furthermore, the osteogenic differentiation capacity was assessed by ALP staining, ARS staining and related gene expressions on the osteogenic differentiation (RUNX2, ALP, and DLX5) after using the P38 signaling pathway inhibitor SB203580.
2.9.Western Blotting.After the cells and newly formed bones were collected, 200 µL RIPA lysate (Beyotime, China) were    2.10.Statistical Analysis.All statistical analyses were conducted using SPSS 21.0, while α level of 0.05 and effect coefficient of 0.90 were defined as the statistical differences.All values were expressed as mean AE standard deviation (SD).
Then the comparisons between two groups were assessed   Stem Cells International by the Student's t-test, whereas multigroup comparisons were analyzed using one-way ANOVA.

Osteogenic Differentiation Ability were Enhanced under the Stimulation by the Inflammatory Factors (S-HuMSCs).
To determine the adipogenic and osteogenic differentiation potential of S-HuMSCs, Figure 1(c) showed that the numbers of lipid droplets were less in the S-HuMSCs than those in the HuMSCs, with significantly lower expressions of PPARγ, Adiponectin, and C/EBPα (P <0:05; Figures 1(g) and 1(i)).Nevertheless, comparing with the HuMSCs, S-HuMSCs could enhance the osteogenic differentiation potential, which was confirmed by the larger staining areas through both the ALP (Figure 1(d)) and ARS methods (Figures 1(e) and 1(f)).Moreover, the expressions of key transcription factors on the osteogenic differentiation (RUNX2, ALP, OPN, DLX5, SP7, OCN, and BMP2) were significantly increased (P <0:05; Figures 1(h) and 1(j)).Simultaneously, we then discussed the roles of IL-32 on the adipogenic and osteogenic differentiation potential.As shown in Figure 4(a), the numbers of lipid droplets in the IL-32 high HuMSCs were significantly less than those in the HuMSCs and NC-HuMSCs groups, with the significant decreases on the gene expressions of PPARγ, Adiponectin, and C/EBPα (P <0:05; Figures 4(b) and 4(c)).Otherwise, the results from ALP, ARS, and mineralized nodules were significantly higher in the IL-32 high HuMSCs than those in the HuMSCs and NC-HuMSCs (P <0:05Þ; Figure 4(d)-4(f )).Meanwhile, the expressions of related key transcriptions on the osteogenic differentiation were significantly increased in the IL-32 high HuMSCs (P <0:05; Figures 4(g) and 4(h)).Furthermore, it was also confirmed by the animal model of cranial bone defect using the Micro-CT (P <0:05; Figure 4 (i)), which was accompanied by the significantly larger areas of new bone formation in the IL-32 high HuMSCs than those in the NC-HuMSCs (P <0:05), with the higher expressions of RUNX2, ALP, BMP2, and DLX5 (P <0:05; Figure 4(k)).the IL-32 high HuMSCs than those in the HuMSCs and NC-HuMSCs (P <0:05), with no significant differences of ERK and JNK pathways, which could be obviously reduced by inhibiting the P38 pathway with SB203580 (P <0:05;

Discussion
Bone healing and regeneration, including the cranial bone, may be affected by the immune microenvironment, vascularization, and so on [21,22].Recently, many researches have proved this phenomenon.For example, Karnes et al. [23] had found that delayed endochondral and intramembranous osteogenesis was shown among the TNFα-receptor-deficient mice.Ishikawa et al. [24] also observed that CCL2-deficient mice could decrease the fracture healing and diminish the callus formation by reducing the infiltration of macrophages, vascularization, and MSCs.Besides, the interactions between  Stem Cells International immune microenvironment and HuMSCs differentiation can regulate the bone regeneration, in which HuMSCs play their roles on regulating the polarization of helper T cells, reducing the activation and proliferation of T cells, inhibiting of M1 macrophages activation, and inducing the generation of M2 macrophages through secreting the cytokines, such as PGE-2, TGF-β1, IL-6, HGF, MCP-1, and COX-2 under the inflammatory state [5][6][7].Then, it had been proposed that MSCs could be pretreated with the proinflammatory cytokines (IFN-γ and TNF-α; S-HuMSCs) to enhance their immunosuppressive properties to promote the bone healing [25][26][27][28].Moreover, Cai et al. [29] found that the immune MSCs were determined to be close with other cell subsets in the cell communication analysis, including the adipogenic-osteogenic differentiation programs.So far, Lu et al. [30] had reported that TNF-α pretreated human AT-MSCs could promote the proliferation and osteogenic differentiation of human primary osteoblasts.However, the limited source of BMSCs and AT-MSCs, and high incidences of related complications affected their wide application on the bone defects [6,31].Therefore, it is particularly important to discuss the roles of S-HuMSCs on the osteogenic differentiation, which was consistent with our conclusion.As a unique cytokine, IL-32 can switch its roles between the proinflammatory and anti-inflammatory programs on many diseases as Crohn's disease, rheumatoid arthritis, chronic obstructive pulmonary disease, ulcerative colitis, psoriasis, atopic dermatitis, and tuberculosis and so on by producing the multiple isoforms through the alternative splicing [32][33][34][35].In addition, other researchers had shown that IL-32 could participate in the airway inflammation by inducing the TNF-α [36].So IL-32 has an important role on promoting inflammatory responses and inducing the differentiation of monocytes into the macrophage-like cells by Stem Cells International arising CD11b, CD14, and CD44, which were the differentiation markers of macrophages [33,35].Moreover, many studies had proved that IL-32 could stimulate the osteogenesis and bone remodeling in vitro and in vivo [37,38].The current study further explored the mechanisms by the S-HuMSCs contributing to the easily osteogenesis.Our previous study suggested that IL-32, GBP2, CD200, PTX3, CHI3L1, and so on were highly expressed in the S-HuMSCs than those in the HuMSCs, in which the induction of IL-32 might be one mechanism to promote the osteogenic differentiation.So our research confirmed that S-HuMSCs could promote the osteogenesis and cranial bone regeneration by upregulating the expressions of IL-32.However, the osteogenesis mechanism of IL-32 is still unclear.Previous studies had shown that IL-32 could augment the productions of proinflammatory cytokines, such as TSLP, IL-1β, TNFα, IL-8, and IL-6, through p38-MAPK, NF-kB, and caspase-1 activation.Besides, the exosomes derived from the MSCs could promote the proliferation of osteoblasts through the P38-MAPK pathway by the osteoinductive ligand BMP2, Wnt signal, parathyroid hormone (PTH) and, etc. [39,40].In summary, IL-32 not only induces the production of inflammatory cytokines, but also activates the typical signaling pathways, mainly the P38-MAPK.In this study, we also verified that the P-P38 in IL-32 high HuMSCs was significantly increased and osteogenic ability was significantly enhanced, which could be decreased when they were treated with SB203580, as a P38-MAPK pathway inhibitor.

Conclusion
In conclusion, our findings have confirmed that S-HuMSCs can enhance the osteogenesis and cranial bone regeneration through promoting IL-32-mediated P38 signaling pathway, which is proved that IL-32 may be a therapeutic target, or a biomarker for the mechanism of bone formation in the treatment of cranial bone injuries.

10
Expressed in the S-HuMSCs.As shown in Figures 2(a) and 2(b), the expressions of IL-32 were specifically higher in the S-HuMSCs than those in the HuMSCs by the single-cell sequencing (P <0:05), which was also verified by RT-PCR (Figure 2(c)) and WB (Figure 2(d); P <0:05).

3. 4 .
IL-32 high HuMSCs Could Promote the Osteogenesis and Cranial Bone Regeneration.As shown in Figures3(a) and 3(b), the transfection efficiency was more than 90%, with no significant differences between the NC-HuMSCs and IL-32 high HuMSCs.Meanwhile, the expressions of IL-32 were much higher in the IL-32 high HuMSCs than those in the HuMSCs and NC-HuMSCs (P <0:05; Figures3(c) and 3(d)), with no changes on the characteristic of purity, selfrenewal, and proliferation (P >0:05; Figure 3(e)-3(h)).

FIGURE 4 :
FIGURE 4: IL-32 high HuMSCs could promote the osteogenesis and cranial bone regeneration: (a) lipid droplets by the oil red o staining; (b) mRNA expression of genes on the adipogenic differentiation; (c) protein levels of PPARγ and C/EBPα; (d) alkaline phosphatase staining; (e) alizarin red staining; (f ) semiquantitative analysis of mineral compounds; (g) mRNA expression of genes on the osteogenic differentiation; (h) protein levels of RUNX2, ALP, BMP2, and DLX5; (i) surgical procedure to create a skull defect; (j) Micro-CT of three-dimensional reconstruction and areas of new cranial bone regeneration; and (k) expressions of osteogenesis related genes in the new cranial bone.All pooled data were represented as mean AE standard deviation (SD, n = 3/group).* P <0:05, * * P <0:01, * * * P <0:001.

TABLE 1 :
Primers of the selected genes.