Wnt11 plays an important role in the osteogenesis of human mesenchymal stem cells in a PHA/FN/ALG composite scaffold: possible treatment for infected bone defect

Abstract

Background: Infected bone defect poses a great challenge for orthopedists because it is difficult to cure. Tissue-engineered bone based on the human mesenchymal stem cells (hMSCs), has currently taken a promising treatment protocol in clinical practice. In a previous study, a porous hydroxyapatite/fibronectin/alginate (PHA/FN/ALG) composite scaffold displayed favorable biological properties as a novel scaffold, which was considered better than single-material scaffolds. In addition, Wnt11 has been demonstrated to play an important role in the development of osteoblasts, but until recently, its role in the osteogenic differentiation of hMSCs in infectious environment remained unclear.

Methods: In this study, we constructed a PHA/FN/ALG composite scaffold with layer-by-layer technology. Furthermore, we also constructed Wnt11-silenced (RNAi) and -overexpressing hMSCs by lentiviral transduction. The gene transduction efficacy was confirmed by quantitative PCR assay and Western blot analysis. Tissue-engineered bone was constructed with hMSCs and PHA/FN/ALG composite scaffolds, and then was implanted into an infected bone defect model for evaluating the osteogenic capacity by quantitative PCR, gross observation, micro-CT and histology analysis.

Results: All those cells showed similar adhesion abilities and proliferation capacities in scaffolds. After tissue-engineered bone implantation, there were high levels of systemic inflammatory factors in vivo, which significantly declined three days after antibiotic therapy. One or two months after implantation, the results of osteogenic-related gene analyses, gross observation, micro-CT and histology consistently showed that the Wnt11 over-expression hMSC group displayed the strongest osteogenesis capacity, whereas the Wnt11-RNAi hMSC group displayed inferior osteogenesis capacity, when compared with the other cell-containing groups. However, the blank control group and the only composite scaffold without cell implantation group both showed extremely weak osteogenesis capacity.

Conclusion: Our results revealed that the Wnt11 gene plays an important role in hMSCs for enhancing the osteogenesis in an infectious environment.

Fig. 1

hMSCs were cultured in vitro and infected with lentiviral vectors targeting Wnt11. a Passage 0 hMSCs show the typical shape of MSCs. b Representative images of hMSCs with lentivirus transduction. c Quantitative PCR assay of the Wnt11 gene expression level in Wnt11 RNAi-expressing or Wnt1-overexpressing hMSCs after transduction; an empty vector expressing enhanced GFP served as a control. d Western blot assay of the Wnt11 protein expression of Wnt11 RNAi-expressing or Wnt1-overexpressing hMSCs after transduction; an empty vector expressing enhanced GFP served as a control. e Quantitative analysis of the relative protein level of Wnt11; results were based on the density and normalized to β-actin. Data presented as mean ± SD. *p <0.05, **p <0.01 and ***p <0.001 compared with empty vector control. Scale bar = 50 μm

Fig. 2

Proliferation and adhesion assay of hMSCs on the surface of PHA/FN/ALG composite scaffolds. a hMSC proliferation assay. b hMSC adhesion assay. c Distribution of DAPI-labeled hMSCs adhered to the scaffolds. Data presented as mean ± SD. ^# p >0.05. Scale bar = 100 μm. hMSC human mesenchymal stem/stromal cell, OD optical density

Fig. 3

PHA was modified with FN and ALG and was transplanted into the infected bone defect after loading with Wnt11-modified MSCs. a Gross appearance of PHA. b Schematic illustration of PHA modified with FN and ALG. c Representative shape of the transfected MSCs. d An infected bone defect (1.0 cm long and 0.5 cm in width) was made for implantation. e PHA/FN/ALG with or without Wnt11-modified MSCs were transplanted into the bone defect site. Scale bar = 50 μm. rFN recombinant fibronectin, PHA porous hydroxyapatite

Fig. 4

Systemic immunological reactions were detected postoperatively, and the serum concentrations of IL-2 a, IL-4 b, IL-6 c, IL-10 d, IFNγ e, and TGF-β1 f were measured at 0, 1, 2, 3, 4, 7, and 14, 28 days after transplantation. No significant differences existed between any two groups at the same time points (p >0.05, n = 3). Data presented as mean ± SD. BCWG blank control without grafts implantation group, ELSG empty lentiviral vectors transduction hMSCs/scaffold group, IFNγ interferon gamma, IL interleukin, OSWG only scaffold without cells implantation group, TGF-β1 transforming growth factor beta-1, WISG Wnt11 RNAi hMSCs/scaffold group, WOSG Wnt11-overexpressing hMSCs/scaffold group

Fig. 5

Expression of relative osteogenic genes in grafts 1 month post operation were tested by quantitative PCR. Data presented as mean ± SD. *p <0.05, **p <0.01, ***p <0.001, ^# p >0.05; n = 3. ELSG empty lentiviral vectors hMSCs/scaffold group, WISG Wnt11 RNAi hMSCs/scaffold group, WOSG Wnt11-overexpressing hMSCs/scaffold group

Fig. 6

Radiographic observation and micro-CT analysis on the osteogenesis of grafts 1 month post implantation. a X-ray observation. b Axial view scanning, coronal view scanning, and three-dimensional reconstruction by micro-CT 1 month post implantation. c Quantitative analysis of osteogenesis in grafts. Data presented as mean ± SD. *p <0.05, **p <0.01, ***p <0.001, ****p <0.0001, ^# p >0.05; n = 3. AV axial view, BCWG blank control without grafts implantation group, BMC bone mineral content, BMD bone mineral density, BV bone volume, BVF bone volume fraction, CV coronal view, 3D three dimensional, ELSG empty lentiviral vectors transduction hMSCs/scaffold group, OSWG only scaffold without cells implantation group, TMC tissue mineral content, TMD tissue mineral density, WISG Wnt11 RNAi hMSCs/scaffold group, WOSG Wnt11-overexpressing hMSCs/scaffold group

Fig. 7

Gross observation of those grafts 1 month after transplantation. a Blank control without grafts implantation group. b Only scaffold without cells implantation group. c Empty lentiviral vectors transduction hMSCs/scaffold group. d Wnt11 RNAi hMSCs/scaffold group. e Wnt11-overexpressing hMSCs/scaffold group

Fig. 8

Histological evaluation of grafts 1 month after implantation. a VG staining; collagen I and trabecular bone shown in blue and red, respectively. b Quantitative analysis based on the area of new bone tissue. Data presented as mean ± SD. **p <0.01, ***p <0.001, ****p <0.0001, ^# p >0.05. BCWG blank control without graft implantation group, ELSG Empty lentiviral vectors hMSCs/scaffold group, OSWG only scaffold without cell implantation group, WISG Wnt11 RNAi hMSCs/scaffold group, WOSG Wnt11-overexpressing hMSCs/scaffold group