Lab Anim Res.  2013 Dec;29(4):196-203. 10.5625/lar.2013.29.4.196.

Bone regeneration of mouse critical-sized calvarial defects with human mesenchymal stem cells in scaffold

• Im JY
• Min WK
• You C
• Kim HO
• Jin HK
• Bae JS

• KMID: 1716214
• DOI: http://doi.org/10.5625/lar.2013.29.4.196

Abstract

Combination of tissue engineering and cell therapy represents a promising approach for bone regeneration. Human mesenchymal stem cells (hMSCs) have properties that include low immunogenicity, high proliferation rate, and multi-differentiation potential; therefore, they are an attractive seeding source for tissue engineering therapy. Here we found that hMSCs with a scaffold did not affect cell viability and osteogenic differentiation. We also investigated regenerative effect of hMSCs with the scaffold in a calvarial bone defect model. Formation of new bone was evaluated by micro-CT, histology and expression of osteogenic markers. The results clearly showed interesting evidence indicating that hMSCs with scaffold increased the formation of new bone and expression of osteogenic markers, compared to the empty and scaffold only groups. Overall, our results suggest that hMSCs with scaffold are suitable for stimulation of intense bone regeneration in critical-sized bone defects.

Keyword

Human mesenchymal stem cells; scaffold; bone regeneration; calvarial bone defect

MeSH Terms

Animals
Bone Regeneration*
Cell Survival
Humans*
Mesenchymal Stromal Cells*
Mice*
Tissue Engineering
Tissue Therapy

Figure

• 

  Figure 1 Viability and differentiation of hMSCs. (A) WST-1 assay was performed on the different scaffolds after 48 h in culture. No differences were observed in cell viability with or without scaffolds. (B,C) Alkaline phosphatase staining, hMSCs with or without scaffold undergo in vitro osteogenic differentiation. Scale bar, 100 mm. Data are expressed as mean±SEM (Student's t-test,*P<0.05 compared with hMSC).

• 

  Figure 2 Differences in osteogenic healing of calvarial defects. (A) Timeline of the experimental design of this study. (B) Four millimeter calvarial defects were created in the parietal bone of nude mice. Treatment groups included empty defects, defects treated with scaffold only, or bone defects treated hMSCs with scaffold (empty group [n=10]; scaffold only group [n=10]; hMSCs + scaffold group [n=10]). Instead, a new layer of woven bone healed the critical-sized defect. Dashed lines encircle the original defect. (C) Micro-CT scanning at eight weeks revealed near complete lack of healing among empty defects. Near healing of hMSCs with scaffold was observed within eight weeks. (D) Quantification of Micro-CT. At eight weeks, healing of defects represented as a fraction of total defect area was quantified by micro-CT images. (ANOVA, Tukey's HSD test, *P<0.005 compared with empty defects group).

• 

  Figure 3 Histologic analysis of bone formation. Serial sections throughout the defect site were created at eight weeks post injury. Defects shown include those empty, those treated with scaffold only immediately after creation of the defect, and those treated with hMSCs with scaffold at eight weeks after creation of the calvarial defect. (A) Stains included aniline blue and pentachrome. (B) At eight weeks, aniline blue-positive bone per 4.0×, 20.0× field was quantified. (C) TRAP staining. TRAP staining was performed for determination of the amount of osteoclasts within the explants from the reconstructed defects. No positive TRAP staining (should be indicated by red color) could be detected within the explants. (D) Osteogenic gene expression by Real-Time PCR at one and two weeks post-injury among hMSC engrafted defects, including hRUNX2, mRUNX2, and mOsteocacin. Data are expressed as mean±SD from four independent experiments (Anova, Tukey's HSD test, *P<0.005 compared with empty defects group). Scale bar, 100 mm, 500 mm.

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