Cell isolation and culture

Mastoid bone marrow was isolated from patients undergoing mastoidectomy.

The study was approved by the Institutional Review Board of the Ajou University School of Medicine (AJIRB-MED-SMP-11-003). Ten patients who underwent mastoidectomies at the Department of Otolaryngology, Ajou University Hospital, Suwon, Republic of Korea, were enrolled in the study. During mastoidectomy, around 1 cc of bone marrow tissues was obtained with a curette from areas without any infection of inflammation and immediately transferred into microtubes filled with Dulbecco’s Modified Eagle’s Medium (DMEM; Gibco-BRL, Grand Island, NY, USA) in the operating room, and was delivered promptly to our laboratory.

Bone marrow was washed with phosphate buffer solution (PBS) containing penicillin and streptomycin, digested with 100 U/mL collagenase type II (GIBCO BRL, Burlington, Ontario, Canada) with low-glucose DMEM, and incubated for 2 h at 37°C.

The stromal fraction was collected by centrifugation at 1200 rpm for 5 min and then passed through a 100-μm cell strainer (BD Falcon, Erembodegem, Belgium) to remove large cell clumps and particles. For cell culture and expansion of hMBMC, the cells were incubated in low-glucose DMEM supplemented with 10% fetal bovine serum (Gibco, Milan, Italy) and 1% penicillin (GenDEPOT, Barker, TX, USA) at 37°C under a 5% CO2

atmosphere. After 24 h, non-adherent cells were removed and the medium was changed.

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When the cells were 50% confluent, they were harvested by trypsinization and subcultured in a fresh medium.

b. Adipose tissue

Adipose tissue was isolated from patients undergoing mastoidectomy. This study was approved by the institutional review board of the Ajou University School of Medicine (AJIRB-MED-SMP-11–002). Twenty patients who underwent mastoidectomies at the Department of Otolaryngology, Ajou University Hospital, Suwon, Republic of Korea, were enrolled in the study. Subcutaneous adipose tissue was removed after postauricular incision and immediately transferred to microtubes filled with Dulbecco’s Modified Eagle’s Medium (DMEM; Gibco-BRL, Grand Island, NY) in the operating room and promptly delivered to our laboratory (Fig. 2). Harvested human adipose tissue was washed with phosphate-buffered saline (PBS) containing penicillin and streptomycin and then digested with 100 U/mL collagenase type I (Gibco BRL, Burlington, ON, Canada) for 2 h at 37°C. Cells were collected by centrifugation for 5 min at 3000 rpm, and the supernatant was discarded. The pellet was resuspended and then passed through a 100-μm cell strainer (BD Falcon, Erembodegem, Belgium) to remove large cell clumps and particles. For culture and expansion of hEASCs, the cells were incubated in low-glucose DMEM supplemented with 10% fetal bovine serum (Gibco, Milan, Italy) and 1%

penicillin and streptomycin (GenDEPOT, Barker, TX) at 37°C under a 5% CO2

atmosphere. After 24 h, non-adherent cells were removed, and the medium was changed.

Upon reaching 50% confluency, the cells were harvested by trypsinization and subcultured in fresh medium.

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Fig. 2. Isolation of mastoid bone marrow and adipose tissue derived from the ear during mastoidectomy. Photograph shows the isolation of mastoid bone marrow and adipose tissue through posterior auriculocephalic sulcus incision.

８ 2. Fluorescent activated cell sorting analysis

The cells were characterized based on their expression of stem cell-specific markers. The cells (1 x 10⁶/mL) were incubated with fluorescein isothiocyanate (FITC)-conjugated CD45, CD29, CD90, and CD105 (BioLegend, San Diego, CA) for 1 h at 4°C.

Cells were analyzed by flow cytometry using BD FACSAriaTM III (BD Biosciences, San Jose, CA). Cells that were not incubated with primary antibodies were used as the negative staining control. To evaluate the maintenance of stemness, the MSCs were analyzed by FACS during each passage.

3. Multi-lineage differentiation of cells

Early-passage cells (passage < 3) were plated at an appropriate density on a culture dish, depending on the differentiation lineage. Five procedures were tested: (1) chondrogenesis [5 x 10⁴ cells/cm²; Chondrogenesis Differentiation Kit, GIBCO, San DMEM media supplemented with 5 μM all trans-retinoic acid (ATRA, Sigma-Aldrich)].

All the differentiation media were replaced every 3–4 days for 14–22 days except the media for neurogenesis which were changed every day for 8 days.

９ 4. Cytochemistry

Chondrocytes were indicated with Alcian blue staining (pH 2.5; Sigma-Aldrich).

The cells were fixed with 4% paraformaldehyde for 20 min and then incubated with 3%

acetic acid at pH 2.5 for 3 min and incubated in Alcian blue solution at pH 2.5 for 30 min.

Osteocytes were observed by mineralization of the extracellular matrix and calcium deposits. The cells were fixed with 80% cold ethanol for 1 h at 4°C. After the cells were rinsed with distilled water (DW), they were incubated with 2% Alizarin Red S solution (Sigma-Aldrich) at pH 5.5 for 10 min. Adipocytes were confirmed by production of lipid droplets using Oil Red O (Sigma-Aldrich). The cells were fixed with 4%

paraformaldehyde for 20 min, overlaid with Oil Red O solution for 6 min, and then washed with 85% propylene glycol and

DW. The samples were stored in PBS at 4°C.

5. Immunofluorescence

Cells were grown on a glass coverslip (Marienfeld, Lauda-Koenigshofen, Germany). The cells were fixed in 4% paraformaldehyde for 20 min and permeabilized using 0.2% Triton-X 100 in PBS for 10 min. After the cells were washed, they were blocked with 1% bovine serum albumin (GenDEPOT, Barker, TX, USA) in PBS and incubated with rabbit anti-Nestin (Abcam, Cambridge, MA, USA), rabbit antineuronal-specific nuclear protein (NeuN; Millipore, Billerica, MA, USA). Secondary anti-rabbit IgG-FITC or Cy3 (Jackson ImmunoResearch Laboratories, West Grove, PA, USA) were applied for 1 h at room temperature. The nuclei were stained with 1 μg/mL

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diamidino-2-phenylindole (Invitrogen, Molecular Probes, Carlsbad, CA, USA) in PBS for 2 min at room temperature. Immunofluorescent cells were imaged using an AxioVision LE 4.5 microscope (Carl Zeiss MicroImaging Inc., Thornwood, NY, USA).

6. Western blot

Proteins were loaded in each lane of a sodium dodecyl sulfate polyacrylamide gel electrophoresis. Primary antibodies against cytokeratin 18 (Abcam, Pic, Cambridge, UK), and β-actin (Cell Signaling Technology, Danvers, MA, USA) were used. The immunoblots

were washed and incubated with secondary antibodies conjugated with horseradish peroxidase (Santa Cruz Biotechnology, Santa Cruz, CA, USA). Immunoblot bands were visualized by enhanced chemiluminescence (GenDEPOT, TX, USA).

7. Histological analysis

The mastoid bulla and cranial bone specimens were fixed in 4%

paraformaldehyde for 24 h and then decalcified in a decalcifying solution (Calci-Clear Rapid; National Diagnostics, Atlanta, GA) for 5 days. The tissues were dehydrated, embedded in paraffin, and sectioned at 10 μm, and the sections were stained with hematoxylin (Youngdong Pharmaceutical Co., Seoul, Korea) and eosin (Muto Pure Chemical Co., Tokyo, Japan) (H & E) and immunohistochemically stained for osteopontin (OPN; Abcam, Cambridge, MA), a marker of mature osteoblasts. For H & E staining, the slides were deparaffinized through two xylene incubations and rehydrated

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through graded washes of ethanol in water. Then, the samples were washed in tap water and stained with hematoxylin and eosin for 3 and 5 min, respectively. For visualization, the slides were mounted with mounting medium (Thermo Fisher Scientific, Miami, FL).

To identify OPN expression, indirect immunohistochemistry was performed using antibodies against OPN. Nonspecific binding was blocked with 1% BSA and 0.2% Triton X-100 in PBS. Sections were incubated overnight at 4°C with the OPN antibody.

Following a wash in PBS, sections were incubated with a biotinylated secondary antibody (Vector Laboratories Inc., Burlingame, CA) for 1 h and visualized using a 3, 30 diaminobenzidine substrate kit (Vector Laboratories Inc., Burlingame, CA). Nuclei were counterstained with hematoxylin. Bright-field microscopy images were obtained using Picture Frame software (Olympus Optical, BX51, Tokyo, Japan)

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

(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