Fall 2008

Scientific Article 2

Recovery Capabilities of Cryopreserved Irradiated Orofacial Bone Marrow Stromal Stem Cells
James Parelli, University of Pennsylvania
President, Zeta Chapter, Psi Omega, School of Dental Medicine

Abstract
Osteoradionecrosis, or tissue death, is a common complication of radiation therapy for head and neck cancer because radiation inevitably destroys normal cells and blood vessels in addition to cancerous cells. Fifty percent of all cancer patients receive radiation therapy, therefore, it clinically important to investigate the mechanisms that induce these iatrogenic side-effects, as well as the ability of specific cell types to recover, both of which may provide insights into improving radiation therapy. This study will evaluate and compare the recovery capacity of irradiated bone marrow stromal cells (BMSCs) from three skeletal sites based on cell proliferation/viability, cell death, p53 expression, and osteogenic property assessed by alkaline phosphatase expression. Two sets of BMSCs will be evaluated: 1) BMSCs cryopreserved for 3 months after irradiation and 2) BMSCs that will be cryopreserved for 3 months, without irradiation, serving as control. The underlying hypothesis is that maxilla/mandible (orofacial bones) BMSCs have better radiation survival ability than iliac crest (axial bone) BMSCs. The clinical significance is that radioresistant BMSCs will be invaluable for stem cell therapies and bone regeneration in osteoradionecrosis.

Background
In various tissues, high doses of radiation have been shown to result in tissue necrosis while lower doses may induce apoptosis. In skeletal tissues, osteoblasts are not as easily destroyed by ionizing radiation as other cell types but their rate of proliferation is significantly impeded [1]. Flow cytometric analysis has shown that radiation induces osteoblast cell cycle arrest at G2/M phase of the cell cycle and consequently increases p53, a transcription factor that regulates the cell cycle [1]. Thus, radiation did not cause osteoblast death but rather induced growth attenuation. Furthermore, irradiation did not induce apoptosis, but sensitized osteoblast cells to apoptogens that were present in the local mineralized matrix. This contrasts with other studies in which apoptotic pathways were directly activated by ionizing radiation [2]. These results indicate that the effects of irradiation are cell-type specific and may affect initiation of osteoradionecrosis following radiotherapy of head and neck cancers. Since BMSCs are osteoprogenitor cells with the ability to form different cell types including osteoblasts, their response to irradiation will have an impact on bone homeostasis and development of osteoradionecrosis. Therefore, this study will evaluate the ability of BMSCs to recover from ionizing radiation.

Orofacial BMSCs isolated from maxilla and mandible have been shown to have superior proliferative and osteogenic properties compared to iliac crest BMSCs [3]. This may relate to their different embryological origins. The craniofacial bones have dual mesodermal and neural crest origins. The maxilla, mandible including alveolar bone, dentin, pulp and periodontal ligament are formed exclusively from neural crest cells, while axial (iliac crest) and appendicular bones are formed from mesoderm. These developmental differences may imply the existence of site-specific properties of progenitor cells in bone marrow that may confer some advantage to orofacial cells with respect to recovery from irradiation. This study will investigate the hypothesized superior recovery capabilities of orofacial BMSCs over iliac crest BMSCs.

Methods
1. Cell Culture
Trabecular bone samples were isolated from 3rd molar surgical sites from the maxilla and mandible, and iliac crest marrow aspirates were also obtained from normal volunteers in a study using Dr. Akintoye’s IRB-approved protocol. BMSCs from maxilla, mandible and iliac crest were cultured in a-MEM supplemented with 20% fetal bovine serum, 100U/ml penicillin, 100 µg/ml streptomycin sulfate and 2mM of glutamine.

2. Cell Irradiation
Maxilla, mandible and iliac crest BMSCs were plated at 1x104 cells/cm2 in T-25 flasks containing a-MEM (as above). Subconfluent cells were irradiated with a Cesium irradiator (Penn School of Medicine) at a dose of 5Gy that is clinically relevant based on therapeutic doses for head and neck cancers. The first set of cells (set I) were immediately centrifuged and cell pellets were resuspended in 1 ml freezing medium (a-MEM, 50 % fetal bovine serum, penicillin (10,000 U/ml), streptomycin sulfate (10,000?g/ ml, and 5% DMSO) and cryopreserved in liquid nitrogen for 3 months until tested. The second set of cells (set II) were non-radiated, but still cryopreserved in liquid nitrogen for 3 months, and served as control.

3. Cell Viability
WST-1 assay: The WST-1 proliferation/viability assay measures the cell’s ability to convert the tetrazolium salt, WST-1 to a colored dye via the activity of its mitochondrial dehydrogenase enzymes. Cells were seeded at 1x104 cells/cm2 in 96 well plates (~3000 cells/well), incubated at 37ºC overnight. After cells incubation with WST-1 reagent, the absorbance was read in spectrophotometer at 450nm.

4. Cell Cycle Analysis
Each cell type was seeded at 104 cells/cm2 in T-175 flasks containing a-MEM (as above). Subconfluent cells were harvested, washed in PBS + 2% FBS and 1 x 106 cells were fixed in ice-cold 70% ethanol for 1 hour at 4°C. Cells were then washed twice with PBS before staining with 50 µg/ml propidium iodide (Sigma-Aldrich, St Louis MO) and RNAse A (Worthington Biochemical Corporation, Lakewood, NJ) dissolved in PBS . Cell cycle analysis was performed using a Becton-Dickinson FacstarPLUS flow cytometer (Core facility at Penn School of Dental Medicine). Propidium iodide fluorescence data was obtained using linear amplification and a minimum of 15000 events were collected.

5. p53 Expression by RT-PCR
Each cell type seeded at 104 cells/cm2 in 60mm dishes containing a-MEM was kept in culture for RNA extraction. Total RNA from sub-confluent cells was isolated with TRIzol® reagent (Invitrogen Life Technologies, Carlsbad, CA) followed by cDNA synthesis using first strand SuperScript™ Double-Stranded cDNA synthesis kit (Invitrogen Life Technologies, Carlsbad, CA). Transcripts for p53 and GAPDH internal control were analyzed on SmartCycler (Cepheid) real-time PCR instruments using a LightCycler–FastStart DNA Master SYBR Green I kit (Roche Applied Science, Indianapolis IN). The optimized primers are presented in Table 1.

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6. Alkaline phosphatase assay
Alkaline phosphatase production was assayed by a method that has been established in Dr. Akintoye’s lab. The cells were cultured and plated. The monolayer was lysed at 4°C with 0.2% cold NP-40 containing 1mM MgCl2 and denatured by repeated freeze-thaw on dry ice. Cells were scraped, sonicated three times in water/wet ice mixture and centrifuged at 800×g for 20 min. Supernatants were assayed for total protein and alkaline phosphatase after 30 min incubation at 37°C using 0.075 M p-nitrophenyl phosphate substrate in 0.05 M barbital buffer, (pH 9.3). The reaction was stopped with 0.1 N NaOH and product measured at absorbance of 405 nm. A standard curve was also plotted using positive controls and standards.

Figure 1. Effect of irradiation and cryopreservation on human bone marrow stromal stem cells (BMSCs). Cryopreserved irradiated BMSCs from the three skeletal sites adhered to culture dish and proliferated with 24hrs. Compared to iliac crest, the maxilla and mandible cells displayed fewer floating dead cells and more spindle-shaped fibroblast morphology.

Figure 2. Proliferation of bone marrow stromal cells subjected to 5 Gy ionizing radiation and cryopreserved was assessed by WST-1 assay. Based on the statistical significance of the data, there was no significant difference in survival of control and cryopreserved-irradiated maxilla and mandible cells. Iliac crest cells showed an apparent increase in metabolic activity after irradiation, suggesting the possible upregulation of cellular activity to compensate for the damage sustained by irradiation and cryopreservation.

Figure 3. Cell cycle analysis of cryopreserved-irradiated cells. Compared with control, the three cell types recovered from the effects of both irradiation and cryopreservation. There were more sub-G0 iliac crest cells (see bar) that indicate possible more apoptotic iliac crest cells compared with maxilla and mandible cells.

Figure 4. Alkaline phosphatase (ALP) assay indicated higher ALP activity in maxilla and mandible cells subjected to 5 Gy ionizing radiation and cryopreservation compared to iliac crest cells, an indication of better osteogenic recovery capacity of maxilla and mandible cells.

Figure 5. Real Time -PCR analysis using optimized p53. Results were normalized using GAPDH control primers. p53 mRNA levels decreased after irradiation in the maxilla and iliac crest cells, but showed no difference in expression in mandible. p53 has been shown to function in varying manners, depending on downstream transcriptions factors, and so further investigation must be conducted to demonstrate the role of p53 in maxilla, mandible, and iliac crest after irradiation and cryopreservation. However, the decreased values of p53 expression in maxilla and iliac crest demonstrates that p53 does play a role in their cell recovery.

Conclusion
The data suggests that bone marrow stromal cell response to radiation is skeletal site-specific, indicating that osteoradionecrosis may have various effects in different locations in the body. Thus, anatomic site-specific radiation susceptibility and recovery capacity may modulate the incidence and pathogenesis of osteoradionecrosis complicating radiotherapy. Of clinical significance to those in the dental profession is the supporting data that orofacial bones, such as those of the maxilla and mandible, show stronger recovery capacity than axial bones (iliac crest) when subject to the harsh conditions of ionizing radiation and cryopreservation, suggesting a possible advantage of tissues derived from specific embryologic germ layer origins. The superior recovery capabilities of orofacial bone marrow stromal cells may open new avenues for therapeutic use in the management of osteoradionecrosis.

Acknowledgements
I would like to thank Dr. Sunday Akintoye and Monika Damek-Poprawa in the Penn Dental Department of Oral Medicine for providing me with the opportunity, insight, and resources to conduct this research study.

References
[1] Szymczyk KH, Shapiro IM, Adams CS. Ionizing radiation sensitizes bone cells to apoptosis. Bone 2004; 34: 148- 156.

[2] Mazur L, Augustynek A, Halicka HD, Deptala A. Induction of apoptosis in bone marrow cells after treatment of mice with WR-2721 and gamma-rays: relationship to the cell cycle. Cell Biol Toxicol 2003; 19(1): 13-27.

[3] Akintoye SO, Lam T, Shi S, Brahim J, Collins MT, and Robey PG. Bone 2006; 38: 758-768.

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