pISSN 1738-2696 · eISSN 2212-5469 http://dx.doi.org/10.1007/s13770-015-0049-8

ORIGINAL ARTICLE

Periimplant Bone Regeneration in Hydroxyapatite Block Grafts with Mesenchymal Stem Cells and Bone Morphogenetic Protein-2 Jee-Hyun Park1, Young-Eun Jung1, Myung-Jin Kim1, Soon Jung Hwang1,2* Department of Oral and Maxillofacial Surgery, Seoul National University Dental Hospital, School of Dentistry, Seoul National University, Seoul, Korea Dental Research Institute, BK 21 Plus, Seoul National University, Seoul, Korea

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Hydroxyapatite (HA) blocks as an alternative material for autogenous onlay bone grafts are regarded as an insufficient substitute for osseointegration of dental implant. In this study, we evaluated the effects of dog mesenchymal stromal cells (dMSCs) with or without bone morphogenetic protein-2 (BMP) on new peri-implant bone formation after HA block graft. In four mandibular bone defects (8×8×6 mm each) in five beagle dogs, dental implants were placed with HA block loaded with autogenous dMSCs with or without BMP-2. Animals were sacrificed at eight weeks, and bone healing was evaluated among four groups consisting of 1) HA alone as a control, 2) HA+dMSCs, 3) HA+BMP-2, and 4) HA+dMSCs+BMP-2. According to histomorphometric evaluation, the MSC+BMP-2 group and the BMP-2 group showed significantly higher bone-implant-contact (BIC) length than the MSC group, while there was no significant difference in new bone formation among the groups. According to micro-CT analysis, bone volume and bone mineral density were significantly higher in the MSC+BMP-2 group compared with the control group (p<0.01 and p<0.05, respectively). BIC was significantly higher in the MSC+BMP-2 group than both the control and MSC groups (p<0.01 and p<0.05, respectively). In conclusion, our results showed that bone regeneration at peri-implant bone defects grafted with HA blocks was significantly increased by dual delivery of MSCs and BMP-2. Conversely, HA blocks with MSC or BMP-2 alone did not allow for efficient peri-implant bone regeneration. Tissue Eng Regen Med 2016;13(1):1-9 Key Words: Dental Implant; Hydroxyapatite; Bone morphogenetic protein-2; Mesenchymal stromal cells; Osteogenesis; Osseointegration

INTRODUCTION Endosseous implant therapy relies on direct implant contact with bone, which is defined as osseointegration [1]. Among the various techniques used to enhance osseointegration of implants placed in bone defects, autogenous bone grafting is considered a predictable and well-documented surgical approach and is accepted as the gold standard for bone regeneration [2]. However, the main disadvantages of autogenous grafts are related to donor site morbidity and graft resorption [3]. Other graft materials such as allogeneic grafts and xenogeneic grafts also have shortcomings such as increased risk of infectious disease transfer and eliciting immunological response [4,5]. As a Received: July 5, 2015 Revised: July 12, 2015 Accepted: July 14, 2015 *Corresponding author: Soon Jung Hwang, Department of Oral and Maxillofacial Surgery, School of Dentistry, Seoul National University, 101 Daehakro, Jongno-gu, Seoul 03080, Korea. Tel: 82-2-2072-3061, Fax: 82-2-766-4948, E-mail: [email protected]

result, there is an increased demand for synthetic bone graft substitutes combined with tissue engineering techniques [6]. Hydroxyapatite (HA) has a good bioaffinity and osteoconductive properties, and can create a positive environment for recruited osteoprogenitor cells to proliferate and differentiate into bone forming cells [7]. HA has been used widely as bone substitute, although its low strength and resorption rate limits its clinical indications. Properties of HA such as sintering temperature, configuration, and pore size also affect its performance [8]. HA with an optimal pore size of 100–400 μm favors bone ingrowth into three-dimensional structures [9]; however the main problem of HA block for alveolar bone augmentation in dentistry is its limited bone forming efficiency in large bone defects due to its osteoconductive properties [10]. There are three basic elements required for bone regeneration by osteogenic stem cells, namely, the ability to differentiate into osteoblasts, regulatory osteoinductive growth factors to induce osteogenesis, and a matrix to provide physical support and direct repair as well as to act as scaffolds for cells and growth © The Korean Tissue Engineering and Regenerative Medicine Society

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Park et al. Hydroxyapatite Block for Onlay Graft with Dental Implant

factors [6]. Mesenchymal stem cells (MSCs) are well-recognized therapeutic cell sources for a variety of clinical applications, and it has been reported that MSCs can act differently according to their origin [11]. Undifferentiated adipose-derived MSCs are predetermined to differentiate to adipose tissue in orthotopic sites compared to bone marrow-derived MSCs [12]. Therefore, bone marrow-derived MSCs are considered one of the main cell sources for bone tissue engineering [13-16]. To enhance osteogenesis, various growth factors related to bone regeneration have been developed, of which bone morphogenic protein (BMP) is regarded as the most powerful osteoinductive growth factor [17]. Indeed, BMP-2 in animal models results has been used successfully to increase levels of new bone formation (NBF) [18,19], and is now clinically used for bone regeneration in the orthopedic and the dental fields [20,21]. The enhancement of bone forming activity of HA using growth factors such as BMP-2 [18,19] or MSCs [15,16] has been described in several studies. However, bone regeneration at periimplant bone defects grafted with HA blocks has not been reported. Moreover, the effects of MSCs and BMP-2 carried by HA block on peri-implant bone regeneration are still unknown. The purpose of the present study was to evaluate bone regeneration at peri-implant bone defects grafted with HA block delivering MSCs and BMP-2 using micro-computed tomography (micro-CT) and histomorphometry in comparison to osteogenesis within HA blocks loaded with either MSCs or BMP-2 alone.

MATERIALS AND METHODS Isolation and culturing of dog mesenchymal stem cells

Heparinized blood (10 mL) was obtained by bone marrow aspiration at the anterior iliac crest of each dog with an 18 gauge bone marrow aspiration needle. dMSCs were isolated and expanded from marrow aspirates using previously published methods [22]. Briefly, dMSCs were prepared by density gradient centrifugation, washed, and plated in Dulbecco’s modified Eagle’s medium-low glucose (DMEM, Welgene Inc., Daegu, Korea) supplemented with 10% fetal bovine serum (FBS, Gibco, Carlsbad, CA, USA) and 100 unit/mL penicillin-streptomycin (Gibco). The cells were then incubated with 5% CO2 at 37°C. After washing off unattached cells over a period of 24–48 hours, the remaining adherent cells were cultured and the medium was replaced every four days. After 10 days individual colonies were collected, isolated, cultured, and expanded. The expanded dMSCs were then replated at an initial density of 1×107 cells/cm2.

Evaluation of the differentiation potential and cell attachment of dMSCs onto HA

The differentiation potential of dMSCs was assessed at pas-

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sage 4 using differentiation-induction medium. To induce osteogenic, adipogenic, and chondrogenic differentiation, cells were seeded onto culture plates with 1.7×104 cells/cm2 and cultured in the respective induction medium for two weeks, during which time the medium was replaced every three days. For osteogenic induction, medium containing 100 nM dexamethasone (Sigma, St. Louis, MO, USA), 10 nM vitamin D (Sigma, St. Louis, MO, USA), 50 μM ascorbic acid (Sigma, St. Louis, MO, USA) and 10 mM β-glycerophosphate (Sigma, St. Louis, MO, USA) was used. To detect bone nodules, calcium deposition was confirmed with von Kossa staining. Adipogenic differentiation was maintained by induction medium containing DMEM highglucose with 10% FBS, 100 nM dexamethasone, 50 μM 3-isobutyl-1-methylxanthine (Sigma, St. Louis, MO, USA), and 10 μg/ mL insulin (Sigma, St. Louis, MO, USA). Cells were rinsed twice with phosphate buffered saline (PBS), fixed with 10% neutral buffered formalin for 30 minutes, and stained with Oil-red O to confirm lipid deposition. To induce chondrogenic differentiation, the cells were cultured in chondrogenic induction medium containing DMEM high-glucose with 10% FBS, 1 mM sodium pyruvate (Sigma, St. Louis, MO, USA), 50 μM L-prolin (Sigma, St. Louis, MO, USA), 500 μM ascorbic acid, and 1% insulintransferrin-sodium selenite (Sigma, St. Louis, MO, USA). The samples were then fixed in 10% formalin and embedded in Tissue-Tek® (OCT compound, Sakura Finetek USA Inc., Torrance, CA, USA), after which they were immediately frozen with (l)N2. The frozen samples were sectioned to a 20 μm-thickness with a cryotome which was cooled with dry-ice. Each frozen sample was then attached in a gelatin coating slide and stored in deep freezer. Next, the sections were dehydrated and stained with 0.1% Toluidine blue for 15 min. A scanning electron microscope (SEM) was used to investigate cell attachment on HA blocks (Bongros®-HA, BioAlpha Inc., Seongnam, Korea). Specifically, dMSCs were cultured in an incubator with a 5% CO2 atmosphere at 37°C for seven days on HA blocks with DMEM supplemented with 10% FBS and 100 unit/mL penicillin-streptomycin, and the medium was replaced on the 4th day.

Preparation of HA blocks: loading of growth factors and dMSCs

The HA blocks used in this study had a porosity of about 70% and an average pore size of 300 μm. After HA blocks were trimmed with a diamond knife to a size of 8×8×6 mm, a single hole with a diameter of 3.5 mm was prepared using a twist drill at the center of the blocks for implant placement. All HA blocks were subsequently washed and sterilized with gamma irradiation prior to use. Blocks to be grafted were loaded with 0.05 mL PBS for the control group and 0.05 mL PBS+dMSCs (3×106 per block) for the MSC group. Next, 25 μg recombinant human

BMP-2 (Novosis®-dent, CGBio Inc., Seongnam, Korea), 0.05 mL PBS for the BMP-2 group, and 25 μg recombinant human BMP2+0.05 mL PBS+dMSCs for the BMP+MSC group were loaded on HA blocks with a micropipette. dMSCs at a concentration of 3×106 per block were suspended in growth-factor admixtures immediately before dropping them onto the HA blocks. The as prepared HA blocks were transplanted into alveolar bone defects 30 minutes after growth factors and/or MSC loading.

Animal surgery

Five adult male beagle dogs (2-year-old) were used for this study. The study protocols were approved by Seoul National University Institutional Animal Care and Use Committee. All the animals were treated and handled in accordance with the “Recommendations for Handling of Laboratory Animals for Biomedical Research” compiled by the Committee on the Safety and Ethical Handling Regulation for Laboratory Experiments at the School of Dentistry at Seoul National University. Animals were maintained in an animal facility at a constant room temperature of 22°C. The animals were fed a soft diet during the first two postoperative weeks; otherwise, there were no restrictions on food. Before surgical procedures the animals were anesthetized with intramuscular injections of tiletamine (Zoletil®, Virbac, Carros, France) 10 mg/kg and xylazine HCL (Rompun®, Bayer, Leverkusen, Germany) 0.4 mg/kg. Animals were then medicated with intramuscular injections of atropine 0.05 mg/kg (atropine sulphate, 0.5 mg/mL, Jeil Pharm. Co., Seoul, Korea), and the first molar and all premolars in the mandible region were extracted. After a healing period of two months, alveolar bone

defects were created and dental implants were installed. A crestal incision was made in the premolar-molar region of the mandible. Full-thickness mucoperiosteal flaps were elevated. Four bone defects in the mandible were prepared surgically with an 8 mm diameter trephine bur, and the residual buccal and lingual bony plates were removed using a fissure bur. This procedure was used to create defects 8 mm in length, 8 mm in width, and 6 mm in height (Fig. 1). Next, titanium dental implants (Ø 3.3×10 mm, Osstem Implant, Seoul, Korea) were installed at the center of the bone defects after the defects were filled with the prepared HA blocks (Bongros®-HA, BioAlpha Inc.), some of which were loaded with rhBMP-2 (CGBio Inc., Seongnam, Korea) and/or autogenous undifferentiated dMSCs cultured from bone marrow aspirates of each dog. Finally, the mucoperiosteal flap was readapted and sutured with resorbable material (Vicryl 4.0, Ethicon). The study consisted of four groups, namely, 1) the control group, HA block alone, 2) the MSC group, HA block+dMSCs, 3) the BMP-2 group, HA block+BMP-2, and 4) the MSC+BMP-2 group, HA block+dMSCs+rhBMP-2 (Fig. 1). During the pre- and postoperative periods, all animals were treated with prophylactic antibiotics (cefazolin sodium 1 g/ vial, 20 mg/kg, Chongkeundang Pharma Co., Seoul, Korea). Prophylaxis was initiated during surgery and continued postoperatively for 3 days. To reduce postoperative pain associated with the surgical procedures, analgesic medication (acetaminophen tablets 15 mg/kg, Daewoo Pharma Co., Seoul, Korea) was administered after surgery for 3 days. All animals were sacrificed eight weeks after implant placement. After sacrifice, mandibles including the four implants and HA blocks were removed after intracarotid artery infusion with 10% neutral buffered for-

Right mandible

Left mandible

M+B B

C M

Defect size (a×b×c)=8×8×6 mm C: PBS

M: PBS+dMSCs

B: PBS+BMP-2

M+B: PBS+dMSCs+BMP-2

Figure 1. Schematic experimental protocol. PBS: phosphate buffered saline, BMP-2: bone morphogenetic protein-2, dMSCs: dog mesenchymal stem cells. www.term.or.kr 3

Park et al. Hydroxyapatite Block for Onlay Graft with Dental Implant

malin for radiological and histological evaluation. The removed mandibles were then sectioned into four smaller blocks each with a width of 1.5 cm. Lastly, the blocks were fixed in 10% neutral buffered formalin.

Micro-CT analysis

Specimens were scanned with a Skyscan 1172 microfocus CT system (Bruker microCT, Kontich, Belgium) with a resolution of 16 μm. Three dimensional scans were taken at an X-ray energy level of 80 kV with a current of 124 μA. The integration time was 316 ms and the stepping rotation angle was 0.4 degrees (450 projections per 180 degrees). The total scanning time per specimen was approximately 15 minutes. Aluminum and copper filters were used to reduce artifacts from the titanium implant. The collected images were 1000×524 pixels in size. Quantitative analysis of bone volume (BV) and bone mineral density (BMD) was performed for volumes of interest (VOIs) with CT analyzer software (ver. 1.8.0.6, Bruker micro CT, Kontich, Belgium). Each VOI consisted of 8 circular cylinders: 1.5 mm in diameter for each VOI at the middle portion of the HA blocks and approximately 1.9 mm in height including 120 slices from the inferior border of the HA block (Fig. 2). The threshold for BV measurements was determined as follows. The HA block was scanned using micro-CT in vitro using the same conditions adopted for in vivo imaging, and the porosity of HA blocks was analyzed using several thresholds, after which the threshold that gave a porosity of 70% was selected. Before BMD measurements, calibrating procedures were performed using BMD calibration phantoms with known BMD values of 0.25 and 0.75, which consisted of fine calcium HA powder uniformly embed-

Figure 2. Quantitative analysis based on micro-CT. VOI for BV and BMD measurements. Each sample was analyzed using 8 circular cylinders with diameters of 1.5 mm at the middle portion of the HA block and 1.9 mm in height from the inferior border of the HA block. micro-CT: micro-computed tomography, VOI: volume of interest, BV: bone volume, BMD: bone mineral density, HA: hydroxyapatite.

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ded in epoxy resin rods. BMD values were expressed in terms of grams per cubic centimeter of calcium HA in distilled water. A zero value for BMD corresponded to the density of distilled water alone (no additional calcium HA), and a value greater than zero corresponded to non-aerated biological tissue. Quantitative analysis of BICs was performed using two-dimensional regions of interest (ROIs) with Image J (National Institutes of Health, Bethesda, MD, USA). Each ROI included the vertically sectioned plane consisting of the implant and HA block. Four vertical planes per sample were sectioned passing through the center of the implant, and the ROI including four threads of the implant from the inferior border of HA block were selected at both sides of each implant (total 8 ROI). MicroCT images corresponding with histologic figures were obtained by adequate vertical cutting of the three dimensional data using a manual procedure. The HA blocks used in this study had a high density in micro-CT images and exhibited a clear geographic outline of their porosity characteristics, which allowed us to identify the corresponding micro-CT images. In comparing micro-CT image with the corresponding histologic section, the brightness and contrast in micro-CT image was adjusted to allow us to effectively distinguish the bone structures from the metal artifacts. Only surfaces adjacent to two continuous pixels with a gray density were included in BIC measurements.

Histomorphometric analysis

After obtaining micro-CT images, the blocks of undecalcified specimens were sectioned through the middle of implants parallel to the long axis of the implants in the mesio-distal direction to include better osseous regeneration from neighboring natural bone surface. This was done because the HA blocks with implants were buccolingually covered only by mucoperiosteal tissue without neighboring natural bone. These specimens were dehydrated in an ascending series of alcohol rinses, embedded in osteo-bed resin (Technovit 7210, Kulzer, Wehrheim, Germany), and polymerized. The resulting tissue blocks with implants were reduced to a final thickness 50 μm with an EXAKT cutting machine (BS-3000N, EXAKT, Norderstedt, Germany), and one section was acquired per bone grafting block. The sections were then stained with a Masson’s Trichrome stain for light microscopic evaluation. Digital images of the stained sections were obtained using an Olympus BX51 transmission and polarized light Axioskop microscope (Olympus Corporation, Tokyo, Japan) equipped with a digital camera (SPOT Insightt, Diagnostic Instrument Inc., Sterling Heights, MI, USA). For bone area (BA), NBF, and BIC measurements, two reference areas and the surfaces of both sides of the implant were selected from each histologic section (Fig. 3). The reference area for BA and NBF measurements consisted of a rectangle 1.5 mm

in width at the middle portion of the HA block and 1.9 mm in height from the inferior border of the HA block. The HA area was included in BA measurements, but was excluded for NBF measurements. The fourth thread from the inferior border of the HA block was used as the reference surface for BIC measurements. The percentage of BIC, BA, and NBF for each ROI was determined using Image J.

Statistical analysis

All data are presented as the mean±standard deviation. Statistical analyses were performed using SPSS 21 (IBM Co., Ar-

monk, NY, USA). Comparison of data between multiple groups was performed using one-way analysis of variance, and the Tukey test was used for post hoc analysis. Results with values of p<0.05 were considered to be statistically significant.

RESULTS dMSC differentiation potential and cell attachment onto HA

To confirm the stem cell properties of dMSCs derived from bone marrow aspirates, their ability to differentiate into osteoblasts, chondroblasts, and adipocytes was evaluated using the corresponding induction medium. When dMSCs were cultured in osteogenic induction medium for two weeks, bone nodules could be clearly observed by von Kossa staining (Fig. 4A). Likewise, dMSCs in adipogenic conditioned medium for two weeks were also successfully induced towards adipogenic differentiation, with lipid droplets confirmed by Oil-red O staining (Fig. 4B). The synthesis of a proteoglycan-rich extracellular matrix by dMSCs cultured in chondrogenic medium was confirmed with Toluidine blue staining (Fig. 4C). Taken together, these results confirmed the stem cell properties of the isolated dMSCs. To investigate the ability of dMSCs to adhere to HA blocks as well as their biocompatibility, HA blocks loaded with dMSCs were observed by SEM seven days after culturing. We confirmed that the cells were well spread and adhered to the HA blocks (Fig. 5).

Histomorphometric analysis

Figure 3. Reference area for BA and NBF measurements used for histomorphometric analysis. BA measurement: two rectangular areas 1.5 mm in width at the middle portion of the HA block and 1.9 mm in height from the inferior border of the HA block. For BIC measurements two surfaces including 4 implant threads from the inferior border of the HA block were selected on both sides of the implant (arrow). BA: bone area, NBF: new bone formation, HA: hydroxyapatite, BIC: bone-to-implant contact.

A  

B  

Wound healing preceded uneventfully at all surgical sites. All operation sites were fully covered with healthy mucosa without soft tissue dehiscence or fistulas. All implants and HA blocks were well fixed at the mandible defect sites. Fig. 6 shows the histologic images obtained after eight weeks of healing. The defects were partially filled with woven bone and HA, as well as ma-

C  

Figure 4. Differentiation of dMSCs into osteogenic, adipogenic and chondrogenic cells. (A) osteogenic differentiation: calcium deposition demonstrated by von Kossa staining. (B) adipogenic differentiation: lipid staining demonstrated by Oil-red O. (C) chondrogenic differentiation: proteoglycan-rich extracellular matrix demonstrated by toluidine blue staining. Original magnification ×400. dMSCs: dog mesenchymal stem cells. www.term.or.kr 5

Park et al. Hydroxyapatite Block for Onlay Graft with Dental Implant

A  

B  

Figure 5. Scanning electron microscopy of dMSCs on HA blocks. Fibroblast-like cells were observed on the scaffold after culturing for 7 days. Original magnifications (A)×100 and (B)×1500. dMSCs: dog mesenchymal stem cells, HA: hydroxyapatite. Table 1. Histomorphometric analysis of specimens

Group

BA (%)

BIC (%)

NBF (%)

Control

51.85±14.01

19.49±17.31

25.25±15.34

MSC

55.04±4.93

9.31±10.76

16.94±3.67

BMP-2

62.04±6.76

42.79±17.38*

35.50±5.98

MSC+BMP-2

65.00±4.96

48.23±9.55

32.25±9.52

†

The values are expressed as mean±SD. Significant difference from the MSC group. *p<0.05, †p<0.01. BA: bone area including hydroxyapatite, BIC: bone-to-implant contact, NBF: new bone formation excluding hydroxyapatite, MSC: mesenchymal stem cell, BMP-2: bone morphogenic protein-2

tured lamella bone, especially in the BMP-2 and MSC+BMP-2 groups. All groups exhibited NBF along the surface of HA, with the NBF tending to begin at the periphery of bone defects, namely, the mesio-distal border and the floor of HA. Increased NBF was observed in the presence of BMP-2 with or without MSCs. In addition, varying degrees of NBF were observed at the gap between the implant and HA blocks resulting from the different diameters of dental implant (3.2) and the preformed hole (3.5 mm), which varied according to treatment group. Importantly, loading with BMP-2 resulted in the gap being predominantly filled with newly formed bone. Quantitative analysis was performed to determine BA, NBF, and BIC, and the BA values were within a small range between 51.85% and 66.04% according to treatment group. Conversely, the BIC values varied widely between groups (9.31% to 48.23%) with the highest values observed for the BMP-2 group and MSC+BMP-2 group. These results implied that peri-implant bone regeneration was strongly enhanced by BMP-2 alone or with combination of MSCs, while bone regeneration at the peripheral area of HA block in conjunction with natural alveolar bone was less influenced by BMP-2 and MSCs. There was no significant difference in BA and NBF among the four groups, even though the mean value of BA and NBF was higher in the

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BMP-2 group and MSC+BMP-2 group than that of the control and MSC groups. However, BIC was significantly higher in the BMP-2 and MSC+BMP-2 group than the MSC group (p<0.01 and p<0.05, respectively) (Table 1, Fig. 7).

Micro-CT analysis

Bone structures were clearly visible on micro-CT scans and a significant contrast was noted between HA and the newly formed bone. On the other hand, while HA could be easily distinguished from metal artifacts, the newly formed bone was not well-defined. Artifacts were present around the surface of titanium implant in all cases. Attempts to remove these artifacts resulted in loss of images of newly formed bone in micro-CT images. The boundary of implant was irregular and not concise due to existing blurred layer along the bone-implant interface. Nevertheless, visual assessment of 2-dimensional CT images was highly consistent with the bony outlines in corresponding histologic sections. In the quantitative analysis for BV, BIC, and BMD of each sample, BV values and BIC values were variable within a small range depending on the specific group as follows: 48.29% and 65.04%, and 28.72% and 54.29%, respectively, with the highest values for BV and BIC noted for the MSC+BMP-2 group. This result implied that MSCs in combination with BMP-2 were effective at inducing bone regeneration not only at the peri-implant area, but also at the peripheral area of the HA block in conjunction with natural alveolar bone. BV was significantly higher in the MSC+BMP-2 group than that of the control group (p<0.01), and BIC in the MSC+BMP-2 group was significantly higher than that of the control (p<0.05) and the MSC group (p<0.01). However, the BMP-2 group did not exhibit a significantly increased BV or BIC compared to the control or the MSC group, even though the mean value of BV and BIC was higher in the BMP-2 group than these two groups.

In addition, BMD was significantly higher in the MSC+BMP-2 group compared to the control group (p<0.05) (Table 2, Fig. 7).

DISCUSSION Alveolar bone defects caused by senile bone atrophy, trauma, or surgical removal of benign or malignant tumors should be augmented when dental implant placements are planned. Autogenous block bone from mandibular ramus or chin can be

B

MSC

Values (%)

Control

A

used efficiently for onlay grafts to increase alveolar bone height or for buccal veneer grafts to widen alveolar bone width. However, donor site morbidity such as prolonged pain, nerve damage, or bone resorption after grafting limits the universal use of autogenous block bone grafts [22]. Thus, in the present study, we investigated HA blocks as an alternative method of bone grafting. Large alveolar bone defects were vertically augmented using HA blocks combined with simultaneous dental implant placement, and bone regeneration was compared among groups depending on the existence of BMP-2 and/or MSCs. The primary aim of this study was to investigate the effects of dMSCs with HA blocks in large bony defects of the canine man-

A  

80 70 60 50 40 30 20 10 0

70

Control MSC BMP MSC+BMP

†

BV BA NBF

*

†

BMP-2

Values (%)

60 50 40 30 20 0

BIC-CT BIC-H *

BMD (g/cm3)

0.9

MSC+BMP

†

*

10

B  

C  

Figure 6. Masson’s Trichrome staining of undecalcified ground sections (50 μm thick) eight weeks after implant placement. Images are in the mesio-distal direction and were observed by light microscopy. Original magnifications (A)×12.5 and (B)×40. MSC: mesenchymal stem cell, BMP-2: bone morphogenic protein-2.

Control MSC BMP MSC+BMP

0.6 0.3 0.0

Control

MSC

BMP-2 MSC+BMP-2

Figure 7. Bone regeneration in micro-CT and histomorphometry eight weeks after implant placement. (A) Bone volume (BV) according to micro-CT analysis, bone area (BA) including HA, and new bone formation (NBF) excluding HA according to histomorphometry, (B) bone-implant-contact (BIC) according to micro-CT and histomorphometry, and (C) bone mineral density (BMD). *p< 0.05, †p<0.01. micro-CT: micro-computed tomography, MSC: mesenchymal stem cell, BMP: bone morphogenic protein, HA: hydroxyapatite. www.term.or.kr 7

Park et al. Hydroxyapatite Block for Onlay Graft with Dental Implant

Table 2. Micro-CT analysis of specimens

Group

BV (%)

BIC (%)

BMD

Control

48.29±6.22

36.02±7.14

0.58±0.08

MSC

53.51±8.06

28.72±6.34

0.64±0.09

BMP-2

55.42±5.75

45.23±9.97

0.66±0.07

MSC+BMP-2

65.04±2.99†

54.29±9.06*‡

0.75±0.03*

The values are expressed as mean±SD. Significant difference from the control group. *p<0.05, †p<0.01; from MSC group, ‡p<0.01. micro-CT: micro-computed tomography, BV: bone volume, BIC: bone-to-implant contact, BMD: bone mineral density, MSC: mesenchymal stem cell, BMP-2: bone morphogenic protein-2

dible on enhancement of NBF. The role of MSCs in repairing tissues has been studied for several decades. MSCs are multipotent cells that can replicate as undifferentiated cells and subsequently differentiate into chondroblasts, myoblasts, adipocytes, nerve-like cells, or osteoblasts in the presence of the appropriate induction factors [23-27]. Various scaffolds for MSCs have been investigated and are now being applied clinically to enhance bone regeneration. Examples of these scaffolds include hydrogels [28], calcium phosphate bone substitutes like HA [29], and xenogenic bone [30-32]. Previous studies demonstrated that MSCs can form primary bone tissue when combined with mineralized three-dimensional scaffolds [33]. However, the present study showed that MSCs alone loaded on HA block did not enhance osteogenesis, and that BIC was actually decreased according the micro-CT and histomorphometrical analysis, which was in contrast with the results of several previous studies [15,26]. The bone defects used in the present study were relatively large, therefore, additional time was necessary for angiogenesis at the peri-implant area distant from the natural alveolar bone, which is a prerequisite factor for the survival and osteogenic function of MSCs. The combination of BMP-2 and MSCs led to the highest level of bone regeneration in the present study, which may have been due to ability of BMP-2 to stimulate angiogenesis in the early healing period [34]. Other factors that may have led to the increased bone formation in the presence of BMP-2 and MSCs included the short loading time of MSCs on the HA blocks before implantation of HA blocks, and non-homogenous loading of MSCs due to the sinking of cells to the bottom side. In other studies that have reported positive effects of the HA scaffold with MSCs on bone formation, the loading time varied from 30 minutes to overnight, with a maximum incubation time of about 14 days before implantation [15,35]. BMP-2 combined with absorbable collagen sponges has been successfully applied to promote localized alveolar ridge or maxillary sinus floor augmentation in humans [21,36]. In the present study, the BMP-2 group exhibited increased osteogenesis in HA blocks, however, the amount of NBF was not significantly different compared to the control and MSC group. Interestingly,

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the bone forming effect of BMP-2 was reinforced with concomitant use of dMSCs. Furthermore, the dual application resulted in significantly higher BV, BIC, and BMDs than the control group. Based on the low bone forming effect in the MSC group and the stimulating effect of BMP-2 on differentiation of MSCs to osteogenic cells, the enhanced bone formation may have been the result of an additive effect rather than a synergic effect. It is desirable for scaffolds used in tissue engineering to have mechanical properties matching that of the host bone. HA blocks have a high compressive strength but low tensile strength, and are therefore fragile. Furthermore, the strength of HA blocks may vary significantly according to the specific manufacturing procedures. On the other hand, collagen sponges are ineffective in providing adequate space for BMP-2-induced bone formation because of their poor physical properties [37]. Indeed, to maintain adequate space for bone regeneration by BMP-2, both HA blocks and xenogenic block bones have been used as scaffolds in combination with BMP-2 [29,31,32]. Importantly, HA alone can be used as a successful carrier for BMP-2 to improve fusion rates, which was demonstrated in a report of rhBMP-2 in a spine fusion animal study [29]. Conversely, bone formation is not increased by BMP-2 when carried with a xenogenic block bone [31,32], and thus HA blocks may be considered as the most favorable scaffold for BMP-2-induced bone formation. However, the HA blocks in the present study tended to break during implant installation in the present study, which decreased the contact of HA blocks with implants. These results suggest that HA blocks mixed with collagen sponges may provide better conditions for onlay grafts, especially at the site of implant placement. In conclusions, bone regeneration at peri-implant bone defects grafted with HA block were significantly enhanced in the presence of both mesenchymal stromal cells and BMP-2. HA blocks with either MSC or BMP-2 alone were not efficient at inducing peri-implant bone regeneration.

Acknowledgements

This work was supported by a grant of the Korea Health 21 R&D Project, Ministry of Health and Welfare, Republic of Korea (A120313). The authors thank Mr. BS Lee for performing histological staining.

Conflicts of Interest

The authors have no financial conflicts of interest.

Ethical Statement

The study protocols were approved by Seoul National University Institutional Animal Care and Use Committee. All animals were treated and handled in accordance with the “Recommenda-

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