J Korean Orthop Assoc.  2018 Apr;53(2):143-151. 10.4055/jkoa.2018.53.2.143.

The Role of Beta-Tricalcium Phosphate-Hydrogel Scaffold and Mesenchymal Stem Cells on Neogenic Bone Formation

• Lee DR
• Kang YK

• KMID: 2410062
• DOI: http://doi.org/10.4055/jkoa.2018.53.2.143

Abstract

PURPOSE
The purpose of this paper was to determine the ability of a mixture consisting of mesenchymal stem cells, beta-tricalcium phosphate β-TCP), and hydrogel, to support cells and form new tissue.
MATERIALS AND METHODS
A composite was produced by adding β-TCP to hydrogel, and mesenchymal stem cells were cultivated in the composite. Then, reverse transcription polymerase chain reaction (RT-PCR) was conducted to measure the level of gene expression for the new bone formation in the cells. Moreover, a composite in which the mesenchymal stem cells were added was injected into the subcutaneous fat of sprague-dawley rats. After four weeks, H&E, Masson trichrome, silver nitrate staining, and osterix immunohistochemical staining were conducted by taking the tissue to evaluate whether the composite supported mesenchymal stem cells and formed new tissue.
RESULTS
By using RT-PCR, we found that the level of gene expression became significantly higher in 3-dimensional gel culture with RUNX2 by 1.26 times, with osteopontin by 1.23 times, transforming growth factor-β by 2.12 times, osterix by 1.07 times, type I collagen by 1.3 times, and fibronectin by 1.3 times. In the animal experiment in which a composite was transplanted into the subcutaneous fat, newly formed tissue was observed. Also, it was found that the composite prevented mesenchymal stem cells from leaving and formed new tissue. Osteogenic differentiation cells in the tissue was observed through osterix immunostaining.
CONCLUSION
It was identified that the composite prevented mesenchymal stem cells dispersal and contributed to the formation of neogenic tissue. Therefore we conclude that the composite plays a role of a scaffold to support the implanted cells and form neogenic tissue more effectively.

Keyword

osteogenesis; mesenchymal stem cells; hydrogel; tricalcium phosphate

MeSH Terms

Animal Experimentation
Collagen Type I
Fibronectins
Gene Expression
Hydrogel
Mesenchymal Stromal Cells*
Osteogenesis*
Osteopontin
Polymerase Chain Reaction
Rats, Sprague-Dawley
Reverse Transcription
Silver Staining
Subcutaneous Fat
Collagen Type I
Fibronectins
Hydrogel
Osteopontin

Figure

• 

  Figure 1 (A) Adipose derived stem cell (ADSC) harvested from adipose tissue after 48 hours of incubation in proliferative medium (×100). (B) ADSC cultured under osteogenic differentiation medium for 2 weeks were stained positively for Alizarin Red S staining, showing mineralization (×100).

• 

  Figure 2 Surface marker expression of adipose derived stem cell (ADSC) after one passage. ADSC cells have positive expression of ADSC markers, including CD44H, CD90.1, and CD106. The cells are free of hematopoietic contamination, as shown by the lack of expression of CD45. Unlabeled cell (negative) control is included for comparison. FITC, fluorescein isothiocyanate; APC, allophycocyanin; PerCP, peridinin chlorophyll protein; PE, phycoerythrin.

• 

  Figure 3 RNA expression relative to glyceraldehyde 3-phosphate dehydrogenase (GAPDH): runt-related transcription factor 2 (RUNX2), osteopontin, transforming growth factor-beta (TGF-β), osterix, type I collagen and fibronectin in the 2-dimensional (2D) plate culture and 3D gel culture at the end of the 3 days. The data are expressed as the mean with standard deviation (n=10) (*p<0.05).

• 

  Figure 4 (A) Macroscopic view of the resected specimen. Subcutaneous injection of the composite from a rat after 4 weeks. (B) Subcutaneous injection of composite from a rat after 4 weeks. (C) Subcutaneous injection of phosphate buffer saline (PBS)+adipose derived stem cell (ADSC) from a rat after 4 weeks. (D) Histological observations: (a) H&E stain of control (PBS+ADSC), (b) H&E stain of composite, (c) Masson trichrome stain of composite and (d) Silver nitrate stain of composite, and (e) Osterix immunohistochemical staining of the composite.

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