In vivo bone formation by human alveolar-bone-derived mesenchymal stem cells obtained during implant osteotomy using biphasic calcium phosphate ceramics or bovine bone as carriers

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In vivo bone formation by human

alveolar-bone-derived mesenchymal stem

cells obtained during implant osteotomy

using biphasic calcium phosphate

ceramics or bovine bone as carriers

Sang-Yeob Oh

Department of Dentistry

In vivo bone formation by human

alveolar-bone-derived mesenchymal stem

cells obtained during implant osteotomy

using biphasic calcium phosphate

ceramics or bovine bone as carriers

Directed by Professor Chang-Sung Kim

The Doctoral Dissertation

submitted to the Department of Dentistry

the Graduate School of Yonsei University

in partial fulfillment of the requirements for the degree of

Ph.D. in Dental Science

Sang-Yeob Oh

This certifies that the Doctoral Dissertation

of Sang-Yeob Oh is approved.

──────────────────

Thesis Supervisor : Chang-Sung Kim

Ui-Won Jung

Seung-Il Shin

Hyun-Seung Shin

In-Woo Cho

The Graduate School

Yonsei University

감사의 글

이 논문이 완성될 수 있도록 부족한 저를 오랫동안 지

도해주시고, 격려해 주신 김창성 교수님께 깊은 감사를

드립니다. 또한 바쁘신 중에도 귀중한 시간을 내주시어

부족한 논문을 살펴주시고 조언해 주신 이중석 교수님,

박정철 교수님께도 감사 드립니다.

아울러 제가 치주학에 입문할 수 있도록 도와주시고,

많은 가르침을 주신 김종관 교수님, 채중규 교수님, 조규

성 교수님, 최성호 교수님, 정의원 교수님, 차재국 교수

님께도 감사의 마음을 전합니다.

연구 기간 동안 많은 도움을 주신 치주과 동기들 및 선,

후배 의국원들과 연구원들께도 감사 드립니다.

마지막으로 언제나 저를 믿어주시고 큰 사랑을 베풀어

주시는 아버지, 어머니와 동생께 깊은 감사를 드리며, 바

쁘신 와중에도 항상 저희 가족에게 많은 도움을 주시는

장인어른, 장모님께도 감사 드립니다. 항상 곁에서 큰 힘

이 되어주는 사랑하는 나의 아내 이진영, 딸 오수민에게

온 마음을 다해 감사의 말을 전합니다.

2017 년 6 월

Table of Contents

List of figures ··· iii

Abstract (English) ··· iv

I. Introduction ··· 1

II. Materials & Methods ··· 4

1. Selection of carrier materials ··· 4

2. Pore size measurement··· 4

3. Isolation of hABMSCs··· 5

4. Cell affinity evaluation ··· 6

5. hABMSC transplantation using MBCP, MBCP-plus, and Bio-Oss

carriers into an ectopic subcutaneous transplantation model ··· 7

6. Histological and immunohistomorphometric analyses of transplanted

samples ··· 7

7. Osteoclast formation by tartrate-resistant acid phosphatase (TRAP)

staining ··· 9

8. Statistical analysis ··· ··· 9

III. Results ··· 10

1. Pore size··· 10

2. Cell-affinity assay··· 10

3 . H i s t o m o r p h o m e t r i c a n a l y s e s a n d h i s t o l o g i c a l

findings··· 11

4. Immunohistological analyses··· 11

5. Immunohistomorphometric analysis··· 12

6. Osteoclast formation··· 13

IV. Discussion ··· 14

V. Conclusion ···18

References ··· 19

Figure Legends ··· 24

Figures ··· 27

Abstract (Korean) ··· 33

List of figures

Figure 1.

Pore sizes of the carriers for human alveolar-bone-derived mesenchymal stem cells (hABMSCs) as measured using micro-computed tomography (CT). MBCP, macroporous biphasic calcium phosphate, MBCP plus, macroporous biphasic calcium phosphate plus.

Figure 2.

Evaluation of the cell affinity of hABMSCs to scaffolds using scanning electron microscopy (SEM) and in vitro assay.

Figure 3.

Ectopic transplantation assays were performed to evaluate the in vivo bone regeneration by hABMSCs loaded onto three different scaffolds.

Figure 4.

Immunohistochemical staining of the ectopic transplantation model.

Figure 5.

Immunohistomorphometric analysis of hABMSCs loaded onto various

carriers using the ectopic transplantation model.

Figure 6.

Tartrate-resistant acid phosphatase (TRAP) staining revealed the presence of multinucleated osteoclasts along the surfaces of the MBCP and MBCP plus particles.

Abstract

In vivo bone formation by human alveolar-bone-derived

mesenchymal stem cells obtained during implant osteotomy

using biphasic calcium phosphate ceramics or bovine bone as

carriers

Sang-Yeob Oh, D.D.S.

Department of Dentistry

The Graduate School, Yonsei University

(Directed by Professor Chang-Sung Kim, D.D.S., M.S.D., PhD.)

Objectives: The aim of this study was to evaluate HA(hydroxyapatite) coated with

different ratios of TCP as a carrier for hABMSCs obtained during implant osteotomy in comparison to slowly-resorbing biomaterial, bovine bone, as a negative control, using in vitro and in vivo experiments.

Materials and methods: Human ABMSCs (hABMSCs) harvested during implant

osteotomy were transplanted using HA/TCP or , bovine bone as carriers in a murine ectopic transplantation model (n=12). Pore size and cell affinity were evaluated in vitro. The area of newly formed bone was analyzed histometrically, the number of osteocytes was counted, and immunohistochemical staining was conducted against

several markers of osteogenesis, including alkaline phosphatase (ALP), runt-related transcription factor 2 (RUNX-2), osteocalcin (OCN), and osteopontin (OPN). Osteoclast formation was evaluated by tartrate-resistant acid phosphatase staining.

Results: MBCP mean pore size diameter was 369.77μm. MBCP plus was 234.13μm

and bovine bone was 238.76μm

. The carrier materials had similar pore sizes.

The

cell affinity assay resulted in a high proportion of cell adhesion (>90%) in all experimental groups. New bone and osteocyte formation were greater in the hABMSC-loaded HA/TCP groups than in the hABMSC-loaded bovine bone group. Positive immunostaining for ALP, RUNX-2, OCN, and OPN was observed with HA/TCP, but only limited expression of osteogenic markers with Bio-Oss. Conversely, there was a minimal osteoclast presence with Bio-Oss, but a significant presence of osteoclasts with both HA/TCP carriers.

Conclusion: Use of HA/TCP ceramics as a cell carrier for hABMSCs resulted in a

significantly greater degree of bone formation in the ectopic transplantation model with than bovine bone. Both types of scaffolds, HA/TCP and bovine bone, showed high stem cell-carrying potential, but the in vivo healing patterns of their complexes with hABMSC could be affected by the microenvironment on the surfaces of the scaffolds.

Keywords: Biphasic calcium phosphate, mesenchymal stem cell, osteogenic

In vivo bone formation by human alveolar-bone-derived

mesenchymal stem cells obtained during implant osteotomy

using biphasic calcium phosphate ceramics or bovine bone as

carriers

Sang-Yeob Oh, D.D.S.

Department of Dentistry

The Graduate School, Yonsei University

(Directed by Professor Chang-Sung Kim, D.D.S., M.S.D., PhD.)

I. Introduction

Mesenchymal stem cells (MSCs) are multipotent cells that can differentiate into many other cells, and are able to regenerate bone, muscle, tendon, and cartilage.(Krampera et al 2006) This has led to the application of MSCs being considered an attractive tissue engineering approach.(Bojic et al 2014; Han et al 2013; Jaiswal et al 1997; Pittenger et al 1999) In particular, stem cells acquired from alveolar bone tissue and alveolar bone marrow have been reported to have great potential for regenerative therapy in the oromaxillofacial area,(Nishimura et al 2012) and Clausen et al.(Clausen et al 2006) reported on the feasibility of using alveolar

bone-derived MSCs (ABMSCs) for cell-based regenerative strategies for bone tissue reconstruction because they can be easily acquired during oral procedures and due to their superior osteogenic potential. We previously demonstrated that ABMSCs can be easily obtained during implant osteotomy using a novel drilling technique under minimal irrigation, and confirmed their outstanding regenerative potential both in vitro and in vivo.(Park et al 2012)

The triad of cells, scaffolds, and growth-stimulating signals is essential for successful tissue engineering.(Chan & Leong 2008) It is necessary to identify the ideal scaffolds in order to deliver the acquired ABMSCs into the desired defect in the oral cavity, since these cells are greatly affected by their immediate microenvironment. Previous studies have shown that MSCs can induce the regeneration of bone defects using biphasic calcium phosphate (BCP) ceramics, absorbable hemostatic gelatin sponge and powder (SPONGOSTAN, Ferrosan Medical Devices, Sydmarken, Denmark), and polymeric materials as stem-cell carriers.(Arinzeh et al 2003; Kadiyala et al 1997; Paganelli et al 2006; Rossi et al 2013) Deproteinized sterilized bovine bone, Bio-Oss (Geistlich, Wolhusen, Switzerland) has also been investigated as a stem-cell carrier in rat calvarium and

maxillary sinus bone augmentation models.(Yu et al 2014a; b) While stem cells possess multilineage potential, it is well established that cell

behaviors are greatly affected by the characteristics of the carrier material upon which they are loaded.(Marklein & Burdick 2010) In our previous study, human periodontal ligament stem cells induced the formation of cementum-like mineralized tissues when

transplanted with hydroxyapatite (HA)/tricalcium phosphate (TCP) particles,(Park et al 2011) while the same stem cells significantly induced the formation of collagen fibers and extracellular matrix when transplanted using hyaluronic acid gel as a cell carrier.(Jung et al 2012) Therefore, the selection of carrier materials should be largely based on the particular carrier characteristics and the desired tissue type for regeneration.

HA/TCP particles are well-established osteoinductive materials and previous studies have shown that the osteogenic potentials of mesenchymal stem cells were significantly induced in ectopic transplantation model.(Park et al 2012; Park et al 2011) The aim of this study was to evaluate HA coated with different ratios of TCP as a carrier for hABMSCs obtained during implant osteotomy in comparison to slowly-resorbing biomaterial, Bio-Oss, as a negative control, using in vitro and in vivo experiments.

II. Materials and methods

Selection of carrier materials

Two different compositions of HA/TCP ceramics – 60% HA/40% TCP (macroporous BCP, MBCP; 500-1000μm; Biomatlante, Vigneux, France) and 20% HA/80% TCP (MBCP plus; 500-1000μm; Biomatlante) – and Bio-Oss (250-1000μm; Geistlich, Wolhusen, Switzerland), were used in particle form as carriers for the hABMSCs in this study.

Pore size measurement

The pore size was measured using micro computed tomography (CT) images by one experienced examiner. Each biomaterial was passively packed into a 1.8 ml cell-freezing vial (n=3 at each group; Cryo Tube, Thermo Fischer Scientific, Waltham, MA, USA), and then the samples were scanned at 18 μm intervals using micro-CT (SkyScan 1072, SkyScan, Aartselaar, Belgium) with settings of X-ray source voltage 70 kVp, current 140 μA, and a 0.5 mm thick aluminium filter. The pixel size was 18 μm, exposure time was 14.7 seconds, the rotation step 0.5°, with a complete rotation over 360°. After scanning, 3D microstructural image data was reconstructed using the

commercial software provided with the equipment (SkyScan, NRecon

version:1.6.6.0). Pore spaces were automatically indicated by the software, with sizes calculated according to the gray values of the threshold in all sectioned images. All

measured data were cumulated with all sectioned images of a sample, and distribution/mean values were calculated.

Isolation of hABMSCs

hABMSCs were isolated and cultured according to a protocol reported in previous studies that demonstrated cell characteristics of mesenchymal stem cells.(Park et al 2012) Briefly, hABMSCs were obtained from the alveolar bone of three donors during dental implant surgery, in accordance with a protocol approved by the Institutional Review Board of Yonsei University (2-2013-0037). A minimal-irrigation technique was employed during implant osteotomy using a slight modification of a previously reported method.(Flanagan 2010) Sequential osteotomies were also executed. The drill speed was limited to 400 rpm (KaVo Intrasurg 300 Plus system, Kavo, Lake Zurich, IL, USA) in the drilling sequence with minimal saline irrigation. The released bone particles were captured in the drill flutes during implant osteotomy. The obtained bone chips were placed into a 50-mL tube and immersed in 20 mL of α-minimal essential medium (α-MEM) containing 15% fetal bovine serum (FBS), 100 U/mL penicillin, 100 μg/mL streptomycin, and amphotericin B (all from Gibco, Invitrogen, Grand Island, NY, USA). The ABMSCs were then immediately isolated using a sequential digestion method with 3 mg/mL collagenase (Wako Pure Chemicals, Osaka, Japan) and 4 mg/mL dispase (Gibco, Invitrogen). They were seeded into 100-mm culture dishes (1×105 cells, five dishes each), incubated for 24 h at 37oC to allow the attachment of adherent cells, and then rinsed twice with

phosphate-buffered saline (PBS) to remove any nonadherent cells. Primary cultures were passed to disperse the colony-forming cells [passage (P)1]. hABMSCs at P3–P5 were used in the experiments. The growth culture medium comprised α-MEM, 15%

FBS, 2 mM L-glutamine, 100 U/mL penicillin, 100 μg/mL streptomycin (Gibco,

Invitrogen), and 100 μmol/L L-ascorbic acid-2-phosphate (Sigma-Aldrich, St. Louis, MO, USA). The isolated cells were characterized as mesenchymal stem cells according to the procedures reported in the previous study (data not shown)(Park et al 2012).

Cell affinity evaluation

The cell attachment potential of hABMSCs to HA/TCP particles and Bio-Oss particles was assessed by subtraction of the number of unattached cells from initially seeded cells. Cells (6x106) at P4 were incubated with each scaffold (80 mg each) in a 1.8 ml-cell freezing vial (Cryo Tube, Thermo Fischer Scientific) overnight, and then the culture media was collected after pipetting several times. The scaffold biomaterials were rinsed three times using additional culture media and pipetting, and these were also collected. The rest of the unattached cells were collected after removal of the particles and treatment with Trypsin-EDTA (Gibco, Invitrogen). The number of remaining unattached cells were counted (CK-40, Olympus Optical, Tokyo, Japan) from all gathered media. Representative scanning electron microscopy (SEM) images of hABMSCs on scaffolds after 24 h of cultivation were taken afterwards (SEM; S-3000N, Hitachi, Tokyo, Japan) to confirm the cell adhesion.

hABMSC transplantation using MBCP, MBCP-plus, and Bio-Oss carriers

into an ectopic subcutaneous transplantation model

The animals were treated under the following three experimental conditions: (1) MBCP and hABMSCs, (2) MBCP plus and hABMSCs, and (3) Bio-Oss and hABMSCs. The hABMSCs (6.0×106) at P4 were mixed with 80 mg of HA/TCP or Bio-Oss particles (80 mg), and the same experimental complexes were transplanted into both left and right dorsal surfaces of 8-week-old immunocompromised mice (total 24 samples), as described previously.(Park et al 2011) Animal selection, management, and surgical procedures followed a protocol approved by the Animal Care and Use Committee of Yonsei Medical Center, Seoul, Korea. The animals were allowed to heal for 8 weeks and then sacrificed.

Histological and immunohistomorphometric analyses of transplanted

samples

Histological and immunohistochemical analyses of transplanted samples were completed according to a previously reported protocol.(Park et al 2012) Transplanted samples (n=24) were fixed with 4% formalin, decalcified with buffered 5% ethylenediaminetetraacetic acid (pH 7.2–7.4). The specimens were embedded in paraffin and cut into 13 sections at a thickness of 6 μm at 18 μm intervals. Three slides were deparaffinized and stained with hematoxylin and eosin, and were

observed using a light microscope (BX-41, Olympus Optical, Tokyo, Japan). Histometric analysis of new bone formation in vivo was performed using Image-Pro software (Media Cybernetics, Silver Spring, MD, USA).

For immunohistochemical analysis, the sections were deparaffinized and epitopes were unmasked using the heat-induced antigen retrieval method with Tris/EDTA. The specimens were immersed in 0.3% hydrogen peroxide to block endogenous peroxidase activity, and then incubated with primary antibodies diluted in PBS (1:200–1:500). A human-specific mitochondrial antibody (mitochondrial ribosomal protein L11, hMito; Abcam, Cambridge, UK) diluted to 1:100 was used to confirm the origin of the human cells, and an antibody raised against proliferating cell nuclear antigen (PCNA) was used as a marker of transplanted cells.(Budke et al 1994) Analysis of markers related to osteogenic tissues was achieved using the following primary antibodies: collagen type I (Col I, Collagen I antibody; Abcam), alkaline phosphatase (ALP, human intestinal ALP; Abcam), runt-related transcription factor 2 (RUNX-2; Novus Biologicals, Littleton, CO, USA), osteocalcin (OCN, OC4-30; Abcam), and osteopontin (OPN, EPR3688; Abcam). A commercially available kit (Zymed SuperPicTure polymer detection kit, Zymed, Invitrogen, Carlsbad, CA, USA) was used to detect the antibodies according to the manufacturer’s protocol.

The slides were mounted and the sections analyzed using the light microscope and digital camera (BX-41, Olympus Optical). Five images of the same dimension and magnification (200×) were taken at four edges and one center area on an image at a magnification 40×, and the area of bone-like tissues and the number of cells

immunopositive for the various antigens was measured histometrically using an automated image-analysis system (Image-Pro Plus, Media Cybernetics).(Jung et al 2012; Song et al 2011) Means and standard deviations were calculated based on the animal means from the two samples of the same subject.

Osteoclast formation by tartrate-resistant acid phosphatase (TRAP)

staining

Three histologic sections from one sample were deparaffinized and stained for TRAP, which is a representative osteoclastogenesis marker, using an acid phosphatase kit (Sigma Chemical, St. Louis, MO, USA). TRAP-positive multinucleated cells with more than three nuclei were considered to be osteoclasts and counted on five selected images by the aforementioned method of regularly chosen areas.

Statistical analysis

For multiple analyses, ANOVA was performed followed by Scheffe’s comparison. The statistical analysis was carried out using SPSS for Windows version 17.0 (SPSS, Chicago, IL, USA). The cutoff for statistical significance was set at P<0.05.

III. Results

Pore size

MBCP, MBCP plus and Bio-Oss particles were investigated using microCT (Fig. 1A) and the macroporosity could be visualized by naked eyes. Apparently, MBCP plus showed the most increased porosity among particles. The distribution of various pore sizes were measured by microCT and reported in figure 1B. MBCP showed overall similar distribution from small to bigger sizes, while Bio-Oss showed increased distribution in smaller pore sizes peaking at 0.124 to 0.195 mm. MBCP plus showed similar pattern to Bio-Oss. Mean pore size diameter were calculated in figure 1C.

Cell-affinity assay

Scanning electron microscopy revealed that round-shaped hABMSCs with short ramifications were evenly attached to all of the scaffold particles (Fig. 2A). At higher magnification, some of the cells on the surface of the carriers appeared to possess podia. In the Bio-Oss group, hABMSCs were sparsely located and naked scaffold surface was more frequently observed than with the HA/TCP carriers. hABMSCs generally appeared to have good affinity with the particles. After overnight culture with the scaffolds, the remaining unattached cells in the growth medium were counted, and the cell affinity was calculated in reverse. The cell affinities in the MBCP and MBCP plus groups were almost 100%, while a

significantly reduced cell affinity was observed in the Bio-Oss group (P<0.05) (Fig. 2B).

Histomorphometric analyses and histological findings

The hABMSCs were seeded onto the scaffolds in an ectopic transplantation model using immunocompromised mice. The mice were allowed to heal for 8 weeks; no significant clinical complications were noted. After the animals were sacrificed, we evaluated the osteogenic activity of hABMSCs in three different carriers (Fig. 3A). At lower magnification, newly formed bone tissues were present along the periphery of the HA/TCP carriers, and several osteocytes were noted inside the mineralized tissues. Along the newly formed bone surface, lining osteoblasts were observed in both the MBCP and MBCP-plus groups. Conversely, only lightly stained areas were observed along the particles in the Bio-Oss group, but minimal bone formation was observed. In addition, there was no osteoblast lining on the surface of the particles of Bio-Oss. Bone regeneration was greater in the hABMSC-loaded MBCP and MBCP plus groups than in the hABMSC-loaded Bio-Oss group (P<0.05), but did not differ significantly between the MBCP and MBCP plus groups (Fig. 3B). The number of osteocytes was also similar in these two groups, but it was significantly lower in the Bio-Oss group (P<0.05; Fig. 3C).

Immunohistological analyses

Immunohistological analyses were performed to identify osteogenic activity at the level of the expression of osteogenesis-related mRNA markers in hABMSCs

loaded onto the three different carriers (Fig. 4). The cells entrapped within the newly formed bone were stained positively for hMito, confirming that the new bone tissues were induced by the transplanted hABMSCs. The osteocytes also were stained positively for PCNA, illustrating the presence of active proliferation by transplanted hABMSCs. Col I makes up more than 90% of the organic matrix of bone and provides the strength, structure, and elasticity of the mature bone tissue.(Hughes et al 2006) Col I positive cells were located along the surface of biomaterials where the new bone is formed. The cells in Bio-Oss group showed very weak reaction to antigens but it appears that the transplanted cells also were attached and proliferated well as well as the other groups.

Immunohistomorphometric analysis

The expressions of mRNA markers related to bone tissue formation, including ALP, RUNX-2, OCN, and OPN, were investigated by immunohistomorphometric analysis to compare the osteogenic potentials of hABMSCs loaded onto the three different scaffolds. (Ducy et al 1996; Ducy et al 1997; Hughes et al 2006; Komori et al 1997; Owen et al 1990; Roach 1994) The numbers of cells that were immunopositive for ALP, RUNX-2, OCN, and OPN were counted (Fig. 5), and there were statistically significant differences observed among groups (Fig. 5B) (P<0.05). In general, the MBCP plus group expressed more positively stained cells for ALP, RUNX-2, OCN, and OPN than other groups.

Osteoclast formation

Several TRAP-positive, multinucleated cells were observed along the surface of the MBCP and MBCP plus particles (Fig. 6A), while there were almost no TRAP-positive cells on the Bio-Oss particles. In particular, small particles in the MBCP plus group were densely surrounded by multinucleated cells, and some exhibited typical resorption pits. The greatest number of cells was observed in the MBCP plus group, and the lowest was found in the Bio-Oss group. The differences between all of the carrier groups were statistically significant (P<0.05).

IV. Discussion

The results of this study show that the use of BCP ceramics as a cell carrier for hABMSCs resulted in a significantly greater degree of bone formation in the ectopic transplantation model with than Bio-Oss. In addition, Bio-Oss was associated with significantly lower induction of osteoclasts along the surface compared to MBCP and MBCP plus. A greater osteogenic differentiation was exhibited by hABMSCs loaded onto MBCP and MBCP plus carriers, as corroborated by immunohistochemical analyses.

It is well established that both Bio-Oss and HA/TCP carriers are recommended for dental application as an alternative to autogenous bone due to their biocompatibility and higher osteoconductive potential.(Araujo et al 2008; Hench & Polak 2002; Mordenfeld et al 2010; Schopper et al 2005; Schwartz et al 1999) However, indications of those previous studies were limited to bone defects in which adjacent bone tissues were able to provide the osteogenic source. The present results--limited osteogenecity with Bio-Oss--were not in accordance with these studies. This could have arisen from the present experimental model of ectopic transplantation, which can provide only limited sources of healing from the surrounding connective tissue. This experimental model is mainly used to evaluate the multipotentiality of cells or osteoinductivity of growth factors by ectopic formation of bone, cartilage, cementum, or ligament from stem cells or growth factor.

The neogenesis in an ectopic site is dependent on the type of related cells, signals, and the characteristics of the carrying scaffold. Our previous study demonstrated different de novo tissue formation by periodontal ligament stem cells with/without bone morphogenetic protein-2 (cementum/periodontal ligament or bone/adipose tissue by the presence of BMP-2).(Lee et al 2014; Song et al 2011) In addition, another of our previous studies found specific collagen formation by stem-cell-carrying hyaluronic acid despite the use of the same periodontal ligament stem cells as in the aforementioned study.(Park et al 2015) The present study also demonstrated different healing patterns dependent on the type of carrying scaffolds: de novo bone formation by hABMSC/BCP but minimal formation by hABMSC/Bio-Oss.

The osteogenic potential of BCP-carrying multipotent stem cells using the ectopic transplantation model has already been noted in many previous in vitro studies, whereas in in vivo studies, they were found to facilitate bone ingrowth.(Saldana et al 2009; Schwartz et al 1999; Sun et al 1997) Arinzhe et al. reported that altering the composition of HA/TCP may influence the amount of bone formation, and demonstrated that when loaded with human MSCs, do novo bone formation in the mouse ectopic model was increased dependently by the proportion of TCP in BCP.(Arinzeh et al 2005) These findings were in line with the present immunohistochemistry result that the MBCP plus group showed significantly more positively stained cells for ALP, RUNX-2, OCN, and OPN, compared to the MBCP as well as the Bio-Oss group. (The MBCP group also showed a greater number of positive cells for the aforementioned osteogenic markers compared to the Bio-Oss

group.) Higher rates of degradation in these carriers would presumably favor the osteogenic potential since the superficial degradation of TCP increases the concentrations of calcium ions along the surfaces, and this calcium-rich microenvironment promotes the differentiation of MSCs, expression of calcium-binding proteins, and calcium incorporation into the extracellular matrix.(Duncan et al 1998; Dvorak et al 2004; Lee et al 2014) In addition, the TRAP staining in the present study also revealed minimal TRAP-positive osteoclast formation with the Bio-Oss carrier, while actively resorbing particles surrounded by numerous osteoclasts were observed with MBCP and MBCP plus (and especially so in the latter). These findings confirm that the microenvironment of scaffolds significantly affected the behavior of hABMSCs and osteoclasts, which are in a coupled relationship.

Bio-Oss is the most widely used and studied biomaterial for dental bone tissue engineering in both clinical and research fields, in which successful results are mainly based on osteoconductivity as well as specific surface characteristics; the dimensions of the grafted biomaterial particles were maintained even at eleven years after surgery,(Mordenfeld et al 2010) and several previous studies found that the surface microstructures of Bio-Oss affected deposition of osteogenic proteins and direct bone formation onto the surfaces.(Araujo et al 2008; Hofman et al 1999; Mladenovic et al 2013; Orsini et al 2005) In this study, hABMSCs were able to attach at very high rates onto the surfaces of all experimental biomaterials (MBCP, 97.89±0.65; MBCP plus, 97.78±1.50; and Bio-Oss, 93.26±2.35), although there were statistically significant

differences between MBCP/MBCP plus and Bio-Oss). However, the present in vivo results showed that the Bio-Oss particles failed to promote hABMSC-induced bone formation as much as MBCP/MBCP plus scaffolds in histology, possibly as a result of different differentiation patterns of the cells in response to the surface characteristics of scaffolds, like isolation of calcium ions from HA/TCP particles. These findings confirm that the microenvironment (including concentrations of calcium ions) of scaffolds significantly affected the behavior of hABMSCs and osteoclasts, which are in a coupled relationship.

V. Conclusion

As hABMSCs carriers, MBCP (20% HA/80% TCP) and MBCP plus (60% HA/40% TCP) exhibited equally and significant osteoinductive potential in the ectopic transplantation model, as evidenced by histomorphometric and immunohistomorphometric analysis, with significantly increased osteoclast formation. While Bio-Oss failed to induce new bone formation when loaded with hABMSCs, it was associated with minimal osteoclast formation along the surface. It therefore appears that both types of scaffolds, BCP and Bio-Oss, showed high stem cell-carrying potential, but the in vivo healing patterns of their complexes with hABMSC could be affected by the microenvironment on the surfaces of the scaffolds, like calcium ions from degraded TCP.

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Figure Legends

Figure 1.

Pore sizes of the carriers for human alveolar-bone-derived mesenchymal stem cells (hABMSCs) as measured using micro-computed tomography (CT). MBCP, macroporous biphasic calcium phosphate, MBCP plus, macroporous biphasic calcium phosphate plus. (A) Reconstructed micro CT images showing different pore composition of three different scaffolds. (B) The distribution of pore sizes was measured and compared. (C) Mean pore diameter was measured in the scaffolds.

Figure 2.

Evaluation of the cell affinity of hABMSCs to scaffolds using scanning electron microscopy (SEM) and in vitro assay. (A) Cells were cultured overnight with each of the following scaffolds: MBCP, MBCP plus, and Bio-Oss, and SEM images were taken. (B) In vitro cell affinity assay revealed that MBCP and MBCP plus exhibited a significantly enhanced cell affinity to hABMSCs in comparison to Bio-Oss (*: P<0.05).

Figure 3

. Ectopic transplantation assays were performed to evaluate the in vivo bone regeneration by hABMSCs loaded onto three different scaffolds. (A) Histologically, newly formed bone tissues were present along the periphery of the HA/TCP carriers, and osteocytes were noted inside the mineralized tissues (arrowheads). However, Bio-Oss was associated with minimal bone formation and no osteoblast lining on the particle. Hematoxylin and eosin (H&E) stain (Upper row: Scale bar= 200 μm, Lower

row: Scale bar= 50 μm). MBCP and MBCP+ were associated with (B) significantly enhanced bone regeneration and (C) the formation of numerous osteocytes within the mineralized tissues (*: P<0.05). However, ABMSCs loaded onto Bio-Oss failed to produce any significant bone formation.

Figure 4.

Immunohistochemical staining of the ectopic transplantation model. (A) hABMSCs loaded onto MBCP particles were associated with newly formed bone tissue and cells stained positively for antibodies (arrowheads) raised against human mitochondria (hMito), proliferating cell nuclear antigen (PCNA), and collagen type I (Col I). hABMSCs loaded onto MBCP plus particles also exhibited bone formation with positively stained cells, comparable with those observed for MBCP. hABMSCs loaded onto Bio-Oss failed to show the presence of positively stained cells (Scale bar = 50 μm).

Figure 5.

Immunohistomorphometric analysis of hABMSCs loaded onto various carriers using the ectopic transplantation model. (A) Cells were stained against the following markers of osteogenesis: ALP, RUNX-2, OCN, and OPN. Positively stained cells were observed in the hABMSCs-loaded MBCP and hABMSCs-loaded MBCP plus groups (arrowheads). Positively stained cells were not observed in Bio-Oss group. (B) The number of positively stained cells were counted and illustrated. MBCP plus group showed statistically significant increase in positively-stained cells (*: P<0.05).

Figure 6.

(A) Tartrate-resistant acid phosphatase (TRAP) staining revealed the presence of multinucleated osteoclasts along the surfaces of the MBCP and MBCP plus particles (arrowheads). (B) There were significantly fewer osteoclasts in the Bio-Oss group when compared than with other carriers (*P<0.05).

Figures

국문요약

임플란트 시술 중 획득한 사람의 치조골 줄기세포의 골형성

능력을 다른 비율의 칼슘 포스페이트와 우골 이식재를

전달체로 사용하여 비교한 연구

<지도교수 김 창 성 > 연세대학교 대학원 치의학과

오 상 엽

사람의 치조골 줄기세포(hABMSCs)는 골재생의 치료에 있어 많은 잠재력을 가지고 있다고 알려져 있다. 많은 연구들을 통하여 사람의 치조골 줄기세포는 임플란트 시술 시 쉽게 채득될 수 있으며 골재생 능력이 있다고 밝혀져 왔다. 따라서, 본 연구는 우골 이식재와 두 가지 다른 비율의 칼슘 포스페이트 이식재를 전달체로 이용하여 사람의 치조골 줄기세포의 골형성 능력을 생체 외, 생체 내에서 비교하기 위함을 목적으로 한다. 사람의 치조골 줄기세포는 12 마리의 쥐 등 부위에 두 가지 다른 비율의 칼슘 포스페이트와 우골이식재를 전달체로 이용하여 이식을 시행하였다. 칼슘 포스페이트와 우골 이식재의 미세구멍크기와 세포 친화력은 생체 외 실험을 통해 측정하였고 신생골의 양은 조직학적으로

측정되었다. Alkaline phosphatase (ALP), runt-related transcription factor 2 (RUNX-2), osteocalcin (OCN)과 osteopontin (OPN)을 이용한 면역조직화학적 검사를 통해 골세포의 양을 측정하였으며 파골세포의 양은 tartrate-resistant acid phosphatase 염색을 통해 시행하였다.

전달체들의 미세구멍크기는 비슷하였고 모두 90 퍼센트 이상의 세포 친화력을 보였다. 신생골과 골세포의 양은 두 칼슘 포스페이트에서 높게 관찰되었으나 우골 이식재는 반대의 결과를 보였다. ALP, RUNX-2, OCN 과 OPN 도 두 칼슘 포스페이트는 높게 관찰되었으나 우골 이식재는 적게 관찰되었으며 파골세포의 발현도 Bio-Oss 가 적게 나타났다. 이상의 연구를 통해, 칼슘 포스페이트와 우골 이식재는 치조골 줄기세포의 전달능력이 높다는 것을 알 수 있었다. 생체 내 실험을 통해서는 전달체의 미세움직임이 사람의 치조골 줄기세포의 재생능력에 영향을 미친다는 사실을 확인할 수 있었다. _____________________________________________________________________ 핵심되는 말: 이상의 칼슘 포스페이트, 중간엽 줄기세포, 골성 분화, 재생