After open cavity mastoidectomy for treatment of chronic otitis media, several complications including recurrent otorrhea, poor hearing aid fit, and the presence of keratin debris may persist. These complications have been reported to occur in approximately 10% of cases, even under the most expert surgical treatment (Palva T et al., 1979). To minimize complications, various autologous tissues and alloplastic materials are frequently used to fill in bone defects. Of these, the musculoperiosteal flap is used most commonly, because it can be manipulated easily and is resistant to infection (Lee HB et al., 2013). However, it is often resorbed, resulting in a large cavity. Other autologous tissues such as cartilage or bone pate are also insufficient to compensate for bone defects (Estrem SA et al., 1999).
In recent years, there has been an increasing interest in stem cell-based tissue engineering (Kagami H et al., 2014), particularly the use of adult MSCs for regenerative medicine, because of the simplicity of isolation and ex vivo expansion of these cells (Nasaf A et al., 2007). Sources of MSCs include bone marrow, adipose tissue, umbilical cord, and amniotic fluid. hMBMCs and hEASCs in particular are easily harvested from the posterior auriculocephalic sulcus during mastoidectomy, without any additional invasive procedures. Therefore, we chose ear-derived mastoid bone marrow and adipose stromal cells as a new source of cells for mastoid bone reconstruction. We first evaluated the regenerative potential of hMBMCs and hEASCs in vitro and subsequently mastoid bone reconstruction in vivo.
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In this study, MSCs could be obtained from most of the patients (9/10, 90%
success rate). Culture was unsuccessful only for 1 patient with a pneumatic mastoid bone.
We think that MSCs could be obtained from most mastoid bones even with the sclerotic type as seen in this study. According to their diagnosis, 6 patients had chronic infection or inflammation and it can be another concern. However, we harvested mastoid bone passage 5, but the expression of CD29 and CD105 began to decrease by passage 6. This finding is consistent with a previous study showing that senescence-like morphology and the number of senescence-associated β-galactosidase-positive cells increased at passage 6 in bone marrow-derived stromal cells (Zheng Y et al., 2016). The authors suggested that p53 plays a critical role in upregulating autophagy during senescence in bone marrow-derived stromal cells (Zheng Y et al., 2016). This relationship between passage number and stem cell potential has been established. Cells at passage 20 showed an expanded cytoplasm and significantly decreased differentiation capacity (Wan Safwaki WK et al., 2011). Although the underlying mechanisms remain unclear, previous reports have suggested that mitochondria, autophagy, genomic stability, and growth factor-mediated changes may affect MSC senescence as the cultivation time and passage number increase
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(Stab BR et al., 2016; Kundrotas G et al., 2016). In addition, changes in stem cell phenotype may differ depending on the MSC origin, which could significantly impact their clinical potential (Meng X et al., 2017). Therefore, stemness must be considered before using MSCs for tissue engineering.
Regarding MSC delivery, a commonly used approach in cell-based therapy is to inject stem cells suspended in saline buffer or culture medium directly into the injured tissues. However, this procedure has not shown satisfactory outcomes; thus, an appropriate supportive 3D scaffold should be applied in cell-based tissue engineering strategies. Scaffolds provide physical and mechanical support, spatial structure, and an adequate biochemical environment for cell behavior (Sivak WN et al., 2014). Another major problem in bone tissue engineering is insufficient oxygen and nutrient supplies, which ultimately cause cell death (Rai B et al., 2010). In previous studies, the porous structure of 3D scaffolds facilitated sufficient oxygen diffusion and nutrient delivery.
We selected PCL as a scaffold for mastoid bulla defect regeneration using hEASCs. PCL is a synthetic, non-toxic, absorbable polymer that can be widely applied in the field of tissue engineering. Porous PCL scaffolds have favorable mechanical properties and are resistant to rapid hydrolysis (Park SY et al., 2016). Jang et al. reported that PCL scaffolds used with human umbilical cord serum(Jang CH et al., 2014) or beta-tricalcium phosphate(Jang CH et al., 2013) led to improved bone formation compared with controls. Our micro-CT imaging and histological results also revealed a significant difference in bone formation between the groups with and without PCL scaffolds.
Interestingly, the positive effect of the scaffolds was much greater for mastoid bulla bone
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formation than for cranial bone formation. This difference may be due to the different environments of these structures. The mastoid bulla does not have any supporting inner structures for cell adhesion in the cavity, and the fluid contents in the bulla can be removed through the Eustachian tube, whereas in the cranial bone defect area, transplanted cells can be supported by the periosteum and supplied with nutrients by adjacent tissues. Therefore, we suggest that a scaffold should be used for stimulating bulla bone formation. Whether PCL scaffolds can exert osteogenic effects on the mastoid bulla bone without the addition of stem cells remains to be elucidated. However, there are some supporting reports that scaffold alone without cell seeding may not have much influence on bone formation. The polyesters (MPEG-(PLLA-co-PGA-co-PCL) scaffold alone showed almost no view of bone-like ingrowths in the bone defects. On the other hand, a significant bone ingrowth was observed when the human dental pulp stem cells were seeded on scaffolds (Kwon DY et al., 2015). And even though bone formation occured with collagen/chitosan/HA scaffold (CCHS) alone, significant difference in bone formation was observed between the CCHS group and the bone marrow-MSC combined with CCHS group (Sun H et al., 2014).
Bone defects of small, non-critical size undergo spontaneous regeneration by recruiting osteoblast-like cells or stem cells from adjacent tissues. These cells are induced to differentiate into osteoblasts by various hormones and growth factors, forming new bone (Marsell R et al., 2011). However, bone defects of a critical size cannot naturally heal without assistance from cells, scaffolds, or growth factors. In this study, we used a commercial ODM to evaluate the effects of osteoinductive growth factors. We observed a
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greater volume and density of regenerated bone in the ODM group than in the non-ODM group. Previous studies showed that Escherichia coli-derived rhBMP-2, BMP-2, and PRP enhanced osteogenesis in mastoid obliteration (Jang CH et al., 2016; Kim SE et al., 2013).
We suggest that ODM is a good option for supplying the nutrients and growth factors necessary for bone formation.
A recent study by Skoloudik et al. (2016) revealed that delivery of stem cell-based biomaterials composed of human bone marrow-derived MSCs (hMSCs), hydroxyapatite, and tissue glue was successful in regenerating temporal bulla bone (Skoloudik et al., 2016). They found that guinea pigs implanted with hMSCs showed a significantly higher ratio of new bone formation than that of non-implanted control animals. Additionally, they evaluated inflammatory processes and angiogenesis but did not find significant differences. However, stem cells can directly or indirectly secrete various immuno- or angiogenic modulatory factors, growth factors, and cytokines that affect their regenerative ability (Gnecchi M et al., 2012).
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