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Published on: December 8, 2016
Osteogenesis with cryopreserved marrow mesenchymal cells
Takafumi Yoshikawa1, Hiroshi Nakajima, Yoshinori Takakura
1Department of Diagnostic Pathology, Nara Medical University, Kashihara City, Nara, Japan. tyoshi@naramed-u.ac.jp
Abstract:
Rat marrow cells were collected from the femurs of 7-week-old male rats (Fischer 344), cultured in 75-cm2 flasks for 10 days, released with trypsin, and then frozen and stored at -196 degrees C in liquid nitrogen. Three months later, the cryopreserved marrow cells were rapidly thawed and cultured in porous hydroxyapatite (HA) blocks in osteogenic medium containing 10 mM sodium beta-glycerophosphate, vitamin C phosphate (82 microg/mL), and 10 nM dexamethasone. After 2 weeks of subculture, cultured cells-HA constructs were subcutaneously implanted into syngeneic rats. The constructs were harvested 2 and 4 weeks postimplantation and examined by histological, biochemical, and genetic analyses. Histological examination showed extensive bone formation in the HA pores. High alkaline phosphatase (ALP) activity and high osteocalcin content were detected in the constructs. Expression of ALP and osteocalcin mRNA was observed at both 2 and 4 weeks. These results indicate that artificial bone prepared with cryopreserved cells had a marked osteogenic capacity.
Insights
Cryopreserved rat marrow cells cultured on hydroxyapatite scaffolds demonstrated significant bone formation potential. This study highlights the osteogenic capacity of cryopreserved cells for artificial bone development.
Area of Science:
- Biomaterials Science
- Regenerative Medicine
- Cell Biology
Background:
- Cryopreservation of cells is crucial for long-term storage and availability.
- Hydroxyapatite (HA) is a well-established biomaterial for bone regeneration.
- Osteogenic differentiation of mesenchymal stem cells is key for bone tissue engineering.
Purpose of the Study:
- To evaluate the osteogenic capacity of cryopreserved rat marrow cells cultured on HA scaffolds.
- To assess the potential of these constructs for artificial bone formation.
- To investigate the feasibility of using cryopreserved cells in bone tissue engineering.
Main Methods:
- Rat marrow cells were cryopreserved and subsequently cultured on porous HA blocks in osteogenic medium.
- Cell-HA constructs were subcutaneously implanted into syngeneic rats.
- Constructs were analyzed histologically, biochemically (alkaline phosphatase, osteocalcin), and genetically (mRNA expression) at 2 and 4 weeks post-implantation.
Main Results:
- Extensive bone formation was observed within the HA pores post-implantation.
- High levels of alkaline phosphatase activity and osteocalcin content were detected.
- Messenger RNA (mRNA) expression for alkaline phosphatase and osteocalcin was confirmed at both time points.
Conclusions:
- Cryopreserved rat marrow cells exhibit significant osteogenic capacity when cultured on HA scaffolds.
- These findings support the use of cryopreserved cells and HA for developing artificial bone.
- The study demonstrates a viable approach for bone tissue engineering using cryopreserved cellular material.
