Bone defects as results of an illness or an accident belong among those with frequent healing complications. One of bad healing wounds are so called critical size bone defects. Such a defect has low capacity to regenerate and also a bad prognosis of tissue function restoration.[1] Implantation of a biocompatible non-cellular collagen type I scaffold with addition of specific inorganic substances naturally occuring naturally in bone extracellular matrix could work as a chanceful alternative therapy approach of these injuries. In this study we examined osteogenic potential of porous scaffolds made from the material Lyopor (Sciteg, a.s.) containing collagen type I, tricalcium phosphate and calcium phosphate in various modifications. Size of pores was measured by scanning electron microscopy. For determination of the level of cellular metabolism an MTS assay based on reduction of substrate by mitochondrial enzymes was used. We also performed rt-PCR in order to quantify relative mRNA expression of osteogenic markers RunX2 and collagen type I. For visualization of bone extracellular protein osteocalcin an immunohistochemical staining was used. Samples were observed using confocal microscopy. After 21, respectively 35 days of cultivation we showed, that any of tested scaffolds does not have a cytotoxic effect on the human mesenchymal stem cells originating from bone marrow. According to the data obtained from rt-PCR we assume the most suitable for osteogenic proteins mRNA expression scaffold composed from collagen type I with hydroxyapatite addition. This carrier also supported proliferation of the cells, which means that its 3D structure was sufficient for mechanical stimulation and support. Strong osteocalcin synthesis was observed on scaffolds containing hydroxyapatite both calcined and non-calcined.
Reference:
[1] Amini, A. R., Laurencin, C. T. & Nukavarapu, S. P. Bone tissue engineering: recent advances and challenges. Crit. Rev. Biomed. Eng. 40, 363–408 (2012).
Acknowledgement: This study was supported by the Ministry of Education Youth and Sports: RP NPU I:LO1309, RP NPU I:LO1508, LQ1601, by the Czech Science Foundation Grant No. 15-15697S, the Ministry of Health: VES 16-28637A, the Czech Science Foundation Grant No. 15-15697S and The Internal Grant Agency of the Ministry of Health of the Czech Republic 17-32285A.
