Animal experimental design - MATERIALS AND METHODS

Ⅱ. MATERIALS AND METHODS

8. Animal experimental design

All animal procedures were approved by the Institutional Animal Care and Use Committee of Ajou University School of Medicine. Thirty-nine male Sprague-Dawley rats (age 8 weeks) were randomly divided into five groups: (1) control group, (2) hEASCs (S group), (3) hEASCs +PCL scaffolds (SP group), (4) hEASCs + ODM (SM group), and (5) hEASCs + PCL scaffolds + ODM (SPM group). The control group was not treated with any reagents or materials. Bone regeneration was monitored by micro-computed tomography (micro-CT) at 2, 4, and 7 months after transplantation. At 7 months post-transplantation, the animals were sacrificed, and cranial bone and tympanic bulla were harvested for histological analyses (Fig. 3A)

１２ 9. PCL 3D scaffolds

To fabricate porous 3D PCL scaffolds, PCL (MW = 80,000, Sigma-Aldrich, Steinheim, Germany) was first dissolved in chloroform (Sigma-Aldrich, Steinheim, Germany). The 5% (w/v) PCL solution was mixed with salt particles (sodium chloride, Amresco, Solon, OH, USA) as a porogen in the range of approximately 100−200 μm.

The mixed solution was poured into a Teflon mold to form the 3D structure. The scaffolds were leached in a distilled water bath for 48 h to eliminate salt particles to form porous 3D structures (Fig. 3B). Finally, the scaffolds were freeze-dried for 72 h.

Scaffolds were sterilized by soaking in 70% ethyl alcohol, then washed in PBS. The fabricated PCL scaffolds with 100−200 µm pore sizes were individually placed into 96-well culture plates and incubated for 15 min with hEASCs (5 × 10⁶ cells/20 μL).

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Fig. 3. Schematic showing the experimental procedures using a hEASCs, PCL scaffold and ODM for bone formation in mastoid bulla and cranial bone defects. (A) Schematic diagram showing the experimental procedures using human ear adipose-derived stromal cells (hEASCs), polycaprolactone (PCL) scaffolds, and osteogenic differentiation medium (ODM) for bone formation in mastoid bulla and cranial bone defects. (B) Three-dimensional (3D) PCL scaffold optical microscopic image (left, scale bar: 1 mm) and SEM image (right, scale bar: 100 μm).

１４ 10. Surgery

The Sprague-Dawley rats were anesthetized by intraperitoneal injection with Zoletil 50 (0.1 cc/100 g; Virbac Laboratoire) and Rompun 2% (0.02 cc/100 g; Bayer Korea). For the mastoid bulla defects, the anterior midline neck skin was incised using a scalpel to expose both sides of the bulla (anterior approach), and 3 × 3-mm holes were made in the bulla using a drill. Then all mucosae inside the bulla were cauterized with 10%

trichloroacetic acid. hEASCs (5 × 10⁶) were transplanted directly into the mastoid bulla in the control group (without ODM) and SM group (with ODM), and hEASC-seeded 3D PCL scaffolds were placed in the bulla cavity in the SP group (without ODM) and SPM group (with ODM). The incisions were closed layer-by-layer. For cranial bone defects, 5

× 5-mm holes were made on the left and right sides of the cranial bone using a trephine bur. The hEASCs, PCL scaffolds (5 × 5 mm), and ODM were transplanted into the defect following the same design as in the bulla defect study.

11. Micro-CT

Radiological examination was performed using Skyscan 1076 (Skyscan, Konitch, Belgium) micro-CT, at a resolution of 36.44 pixels and exposure time of 300 ms, with an energy source of 35 kV and current of 170 mA. Each bulla and cranial bone, consisting of 622 slices on average, were scanned. The CT images were processed using MIMICS 16.0 3D imaging software (Materialise’s Interactive Medical Image Control System, Leuven, Belgium). Newly formed bone was identified by assigning a threshold for total bone mineral content. For quantitative comparisons among groups, we

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calculated bone volume (BV, mm³) and BV/total volume (BV/TV, %) from the micro-CT images.

12. Statistical analysis

Statistical comparisons between two groups were calculated by the Mann-Whitney U test and comparisons among three groups by the Kruskal-Wallis test using SPSS software version 12.0 (SPSS, Inc., Chicago, IL, USA). P value of P < 0.05 was considered significant.

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III. RESULTS

1. Clinical profiles of the patients

The characteristics of the patients are summarized in Table 1. The average age of the patients was 32.8 ± 22.6 years (range; 3 - 66 years). Their diagnoses were chronic otitis media (n = 5), cholesteatoma (n = 1), sensorineural hearing loss (n = 3) requiring cochlear implantation, or vestibular schwannoma needing surgery via the translabyrinthine approach. The degree of mastoid pneumatization was divided into 3 types (pneumatic, sclerotic, and diploic) according to the different amounts of pneumatized air cells. Five (50%) patients showed the pneumatized type, while the others (50%) revealed the sclerotic variant. Culture was unsuccessful only for 1 patient (10%) with a pneumatized mastoid bone.

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Table 1. Clinical characteristics of patients.

Case Age

(years)

Sex Diagnosis Site Operation Name Harvest

Method

COM = chronic otitis media; Chole = cholesteatoma; SNHL = sensorineural hearing loss;

VS = vestibular schwannoma; R= right; L = left; OCM = open cavity mastoidectomy; CI

= cochlear implantation

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2. Characterization of mastoid bone marrow-derived cells

During passage 0, hMBMCs showed characteristic neurosphere-like clusters of cells that changed into a much more stretched form resembling fibroblasts during subsequent passaging over 60 days (passage 3) (Fig. 4). The characteristics of the isolated cells were first evaluated by flow cytometry using CD markers to identify potential stem cells. CD105 (endoglin) is a member of the transforming growth factor beta family, which has been linked with multi-lineage differentiation capacity (Alev C et al., 2010), while CD29 (integrin beta 1) is responsible for mediating cell-to-cell adhesion and is involved in cell growth, differentiation, migration, and death (Langan RC et al., 2012).

CD90 (Thy-1) is expressed in colony forming cells with high proliferative potential and is also used to characterize stem cell populations. In contrast, CD45 is expressed in hematopoietic stem cells (Kucia M et al., 2005).In our study, as shown in Fig 4B, populations of cells yielded positive results for CD90, CD105, and CD29 (99.52%, 99.88%, and 85.49%, respectively) and negative results for CD45 (0.97%) at passage 1.

These data indicated that adherent cells derived from mastoid bones display an MSC phenotype.

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Fig. 4. Characteristics of hMBMCs. Phase-contrast image on day 5, passage 0 (A), and on day 60, passage 3 (B). (C) FACS analysis for MSC-specific surface markers (CD45, CD29, CD90, and CD105) at passage 1.

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3. Maintenance of stem cell phenotype in mastoid bone marrow-derived cells Expansion and maintenance of stem cell phenotype in vitro are required for clinical autologous cell transplantation. However, the efficiency or changes in hMBMCs during prolonged culture have not yet been studied well. The multipotent ability of MSCs gradually decreases depending on the passage number (Kucia M et al., 2005). To evaluate the stemness of the hMBMCs, we cultured hMBMCs during passage 12 and assessed the levels of MSC immune-phenotypes by using flow cytometry. Interestingly, hMBMCs stably maintained their stem cell phenotype up to passage 4 (Fig. 5). However, after passage 4, we observed a gradual decrease in the expression of CD90, CD105, and CD29 (Fig. 5).

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Fig. 5. Maintenance analysis of the hMBMC stemness at passages 2, 4, 6, 8, 10, and 12. To FITC:fluorescein isothiocyanate.

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4. Differentiation of mastoid-derived cells into the chondrogenic, osteogenic, adipogenic, neurogenic and epithelial Lineages

To induce hMBMCs differentiation, we used a culture medium containing specific supplements. On incubation with an adipogenic induction medium for 22 days, the morphologic features of the hMBMCs were changed from typical fibroblast-shaped cells to polygonal cells containing droplets in the cytoplasm (Fig. 6A). To observe the lipid-droplet accumulation more clearly, hMBMCs were stained with Oil Red O. Oil Red O-positive lipid droplets were found in all the cells. In contrast, Oil Red O-positive cells were not detected in the control hMBMCs (Fig. 6B). Under the chondrogenic condition, hMBMCs acquired a cuboidal morphology (Fig. 6A). Alcian blue (pH 2.5) was used to demonstrate the presence of acidic mucopolysaccharides and proteoglycans, which are useful markers of mature chondrocytes (Mitchell JB et al., 2006). With commercial chondrogenic differentiation media, high efficiency was noted for bluish staining (acidic mucosubstances), but no staining was observed in the control hMBMCs (Fig. 6B). Next, we observed robust growth and cluster formations after osteogenic induction. Alizarin Red S staining showed large amounts of calcium deposits in hMBMCs, but not in the control cells (Fig. 6B). In the case of neuronal differentiation, we found that hMBMCs stopped proliferation and entered the differentiated state after neuronal induction as opposed to the results seen for the control hMBMCs. Moreover, the cells exhibited a contracted cytoplasm and spherical cell bodies. Neuronal differentiation was analyzed using an immature neuronal marker, TUJ1 (beta-tubulin 3), and a mature neuronal marker, NeuN. As shown in Fig. 6C, the TUJ1 and NeuN markers were expressed in a small

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percentage of cells. Finally, we tried to induce epithelial cell differentiation in the presence of retinoic acid. Retinoic acid has been reported to induce differentiation toward the epithelial lineage (Palva T et al., 1979). After 22 days, in the presence of a retinoic acid-containing medium, the hMBMCs exhibited a change in morphological features.

Western blot analysis was performed to quantify the epithelial cell marker, cytokeratin 18.

As shown in Fig 6D, the cytokeratin 18 level significantly increased in the retinoic acid-containing medium.

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Fig. 6. Multiple differentiation potential of hMBMCs. (A) Phase-contrast images obtained on days 1, 8, 15, and 22 after differentiation induction. (B) Specific phenotype images with Oil red O (for the adipogenic lineage, upper), Alcian blue (for the

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chondrogenic lineage, middle) and Alizarin Red S (for the osteogenic lineage, lower) staining. (C) Immunocytochemical co-staining with TUJ1 (red) and NeuN (green, arrows) after 8 days of neuronal induction. The picture on the left side shows the undifferentiated hMBMCs. (D) Western blot analysis of cytokeratin 18 after epithelial cell induction. β-actin was used as a loading control.

２６ 5. Characterization of hEASCs

The hEASCs isolated from patients who underwent mastoidectomy were characterized by flow cytometry for several cell surface markers, including CD45 for hematopoietic cells and CD90, CD105, and CD29 for MSCs. We found that hEASCs expressed CD90, CD105, and CD29 (100%, 100%, and 97.4%, respectively) and were negative for CD45 (0.7%) at passage 0 (Fig. 7A). In vitro differentiation assays indicated that the hEASCs induced chondrogenic, osteogenic, adipogenic, and neurogenic differentiation as shown by alcian blue staining, alizarin red S staining, oil red O staining, and NeuN immunofluorescence, respectively (Fig. 7B). hEASCs isolated from behind the ear thus show potential as a source of cells for bone reconstruction. Studies have shown that the potential and maintenance of stem cells may change depending on their origin over a progression of cell passages. However, not all researchers have used the same isolation and passage conditions for adult stem cells. Therefore, we evaluated the stemness of hEASCs. The hEASCs stably maintained their stem cell phenotype up to passage 4. However, after passage 5, we observed a gradual decrease in the expression of CD105 and CD29 (Fig. 7C). Because serial passaging of stem cells influences their phenotype, stem cells must be cultured with care.

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Fig. 7. Characteristics and maintenance analysis of the stemness surface markers and differentiation capacities of hEASCs. (A) Representative fluorescence-activated cell sorting (FACS) analysis showed positive for mesenchymal stem cell (MSC) specific surface markers (CD45, CD90, CD105 and CD29) at passage 1. (B) Multipotent differentiation potential of hEASCs. Specific treatments induced hEASCs to undergo chondrogenic (Alcian blue staining), osteogenic (Alizarin red S staining), adipogenic (oil red O staining), and neurogenic (immunocytochemical staining with NeuN, FITC) differentiation. An increased numbers of differentiated cells were observed in the hEASC population. (C) Maintenance analysis of hEASC stemness at passages 0‒7. The hEASCs consistently maintained their stem cell phenotype up to passage 4. After passage 5, gradual decreases of CD105 and CD29 were observed. FITC: fluorescein isothiocyanate.

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6. Bone formation in rat mastoid bulla and cranial bone defects

On CT images taken 2 and 4 months after surgery, the SP (hEASCs + PCL) and SPM groups (hEASCs + PCL + ODM) showed significant new bone formation (Fig. 8A).

In contrast, the groups without PCL scaffolds (control, S, and SM groups) did not show any regenerated bone. Histological staining and OPN immunohistochemistry also showed new bone formation at the periphery of the bulla defects in the groups transplanted with PCL scaffolds (Fig. 8B, C). In addition, the cranial bone defects of the SM (hEASCs + ODM) and SP groups (hEASCs + PCL) showed new bone formation, but there were no observable effects of ODM (Fig. 9). These results indicate that the PCL scaffold was essential for new bone formation in the mastoid bulla defects, but not in the cranial bone defects.

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Fig. 8. In vivo osteogenic effects of hEASCs, PCL scaffold and ODM. Coronal plane computed tomography (CT) images (A), H&E staining (B), and immunohistochemical analysis of osteopontin (OPN; C) of the bulla cavity at 2 and 4 months after mastoid bulla obliteration in the animal study. The hEASCs + PCL and hEASC + PCL + ODM groups showed significant bone formation compared with other experimental groups. The yellow and black arrows indicate rat mastoid bulla defects. Scale bars: 2 mm.

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Fig. 9. Evaluation of in vivo osteogenic effects of hEASCs, PCL scaffold and ODM.

Coronal plane computed tomography (CT) images (A), H&E staining (1.25X (B), 20X (C) of the cranial bone at 2 and 4 months after bone defects in the animal study. Scale bars: 2 mm.

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7. ODM synergistically enhances cell/scaffold-based bone formation

For quantitative evaluation of bone mineral density and the effects of ODM on the SP group (hEASCs + PCL), we performed micro-CT and 3D-image conversion using the MIMICS 16.0 software on new bone defects in vivo, including the control, SP (hEASCs + PCL), and SPM (hEASCs + PCL + ODM) groups. As shown in the 3D images (Fig.

10C), bone formation in the SP and SPM groups occurred along the periphery of the bulla defects and grew into the scaffolds (Fig. 10A, C). In the control group, there was no evidence of new bone formation at the defect. After 7 months, the BV was 25.8 ± 1.0 mm3 in the control, 34.2 ± 4.7 mm3 in the SP group, and 43.3 ± 2.5 mm3 in the SPM group. The BV/TV was 18.0 ± 1.8% in the control, 23.2 ± 2.6% in the SP group, and 27.8

± 4.2% in the SPM group (Fig. 10D). There were significant differences among the three groups (P < 0.05). This suggests that the hEASCs and PCL scaffold have potential for osteogenesis, and ODM may greatly enhance osteogenic induction.

The cranial bone also showed increased BV in the SP and SPM groups compared with the control group. However, ODM did not induce any significant differences (Fig. 11).

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Fig. 10. Micro-CT and 3D reconstruction images of tympanic bulla bone formation.

(A) Micro-CT analysis of tympanic bulla in the control, hEASCs + PCL, and hEASCs + PCL + ODM groups at 5 and 7 months. (B) Representative micro-CT 3D reconstructed images of the bulla cavity bone. Quantitative morphometric analysis of bone volume (C) and bone volume/total volume ratio (D) in the bone defect as assessed by micro-CT. Area within the yellow box in the 3D image (above) is viewed at a higher magnification (below). The yellow and black arrows indicate rat mastoid bulla defects.

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Fig. 11. Micro-CT and 3D reconstruction images of cranial bone formation. (A) Micro-CT analysis of cranial bone in the control, hEASCs + PCL, and hEASCs + PCL + ODM groups at 5 and 7 months. (B) Representative micro-CT 3D reconstructed images of cranial bone. Quantitative morphometric analysis of bone volume (C) and bone volume/total volume ratio (D) in the bone defect as assessed by micro-CT. The yellow and black arrows indicate rat cranial bone defects.

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IV. DISCUSSION

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

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