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Basic and Clinical Research

Clinical Study of Bone Regeneration by Conditioned Medium From Mesenchymal Stem Cells After Maxillary Sinus Floor Elevation

Katagiri, Wataru DDS, PhD*; Watanabe, Junna DDS; Toyama, Naoto DDS; Osugi, Masashi DDS, PhD; Sakaguchi, Kohei DDS; Hibi, Hideharu DDS, PhD§

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doi: 10.1097/ID.0000000000000618
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Prosthetic rehabilitation of the atrophic edentulous maxilla using implants is a challenge because of insufficient alveolar bone volume, especially in the posterior area. Maxillary sinus floor elevation (SFE) techniques have been used to improve the bone volume of the atrophic posterior maxilla. Clinical studies of maxillary SFE and several bone grafting procedures, including grafting with biomaterials, have been conducted to allow more predictable and strategic implant-supported prostheses that restore both function and esthetics. Autogenous bone grafts have long been used because of their osteoinductive and osteoconductive properties and immunogenic compatibility. However, the use of autogenous bone grafts has limitations such as donor site morbidity, insufficient quantity, and bone resorption in the recipient after a long period of healing.1

A variety of biomaterials have been introduced as substitutes for autogenous bone grafts. Furthermore, local delivery of growth factors has been shown to enhance bone formation. Platelet-rich plasma (PRP), a cocktail of growth factors such as platelet-derived growth factor (PDGF) and transforming growth factor (TGF)-β, is an effective bone regenerative agent.2 Bone morphogenetic protein (BMP)-2 has been used widely with good efficacy in maxillary SFE, although adverse events including facial and oral edema have been reported.3–5

Along with changes in materials, the potential to apply concepts from the arenas of tissue engineering and regenerative medicine, such as bone regenerative medicine for implant site development has been demonstrated in the past 2 decades. Applications of bone-tissue engineering and regenerative medicine have been extensively studied and have become a reality in daily clinical practice. One of the major sources of cells for bone-tissue engineering and regenerative medicine is mesenchymal stem cells (MSCs) from bone marrow. “Tissue-engineered bone,” in which autogenous human MSCs are combined with PRP, has been used as an alternative to biomaterials, and predictable good outcomes have been reported.6,7 However, there are currently several clinical problems in stem cell-based medicine. Viability of the implanted cells has been low in some studies, and cell-handling processes, including quality and safety management, are complicated and expensive.8,9

Recent studies have shown that implanted cells may contribute to tissue regeneration through their pluripotency as well as their paracrine effects on host cells.10,11 The secretome of MSCs contains many growth factors, such as insulin growth factor (IGF)-1, vascular endothelial growth factor (VEGF), and TGF-β1, that can affect the migration, angiogenesis, and osteogenic differentiation of endogenous MSCs, and serum-free conditioned medium from MSCs (MSC-CM) includes such growth factors and has great potential for bone and periodontal tissue regeneration.12–15 Our previous study showed that a mixture of MSC-CM and beta-tricalcium phosphate (β-TCP) enhanced the early phase of bone regeneration after maxillary SFE in rabbits.16

Based on these preclinical experimental studies, we planned to perform a clinical study using MSC-CM for bone regeneration after maxillary SFE. This preliminary clinical study was conducted to evaluate the safety of use of MSC-CM before a phase I clinical study.

Materials and methods

Cell Culture and Preparation of MSC-CM

Human MSCs were purchased from Lonza Inc. (Walkersville, MD) and cultured in MSCBM Mesenchymal Stem Cell Basal Medium (Lonza Inc.) with MSCGM Mesenchymal Stem Cell Growth Medium SingleQuots Supplements and Growth Factors (Lonza Inc.). Cells of the third passage were used in this study. Cells were maintained at 37°C in 5% CO2/95% air.

When the human MSCs reached 70% confluency, the medium was refreshed with 10 mL of serum-free Dulbecco's modified Eagle's Medium (Gibco; Thermo Fisher Scientific, Waltham, MA) containing antibiotics (penicillin G, 100 units/mL; streptomycin, 100 μg/mL; and amphotericin B, 0.25 μg/mL; Thermo Fisher Scientific). The cell culture medium was collected after incubation of cells for 48 hours. For clinical use, the cell culture medium was concentrated and stored. Briefly, the cell culture medium was centrifuged for 3 minutes at 407g. The supernatants were collected and centrifuged for 3 minutes at 4°C and 1630g to remove cells, and passed through a filter of 0.22 μm pores (Millex GP Filter Unit; Merck Millipore Ltd., Darmstadt, Germany). This medium was defined as MSC-CM.

Five milliliters of MSC-CM with 45 mL of 100% ethanol and incubated at −20°C for 1 hour. The mixture was centrifuged for 15 minutes at 4°C and 24,229g, and the supernatant was discarded. The precipitate was resuspended in cold 90% ethanol and centrifuged for 15 minutes at 4°C and 24,229g. The final precipitate was frozen at −80°C, lyophilized, and stored at −80°C until use.

Before clinical use, MSC-CM was examined not only for contamination with bacteria, fungi, or mycoplasmas but also for infection with viruses including hepatitis B and C viruses, human immunodeficiency virus, and human T-cell leukemia virus.

Patient Selection

Six partially edentulous patients who were diagnosed as needing maxillary SFE and bone graft were enrolled in this study. The criteria for application of these procedures are <5 mm of residual bone from the sinus floor to the alveolar ridge, defects of dentition in the posterior maxilla, and proposed rehabilitation with an implant-supported prosthesis. All patients were healthy and free from any disease that could have influenced the outcome of this study (eg, diabetes, autoimmune disease, bone disease, endocrine disease). Each patient provided written informed consent after receiving detailed information about the surgical procedures, graft materials, alternative treatments, and uncertainties associated with using a new method. Two of 6 patients who declined to use MSC-CM were grouped as the β-TCP group. The ethics committee of Nagoya University Hospital approved this research protocol (No. 3437).

Preparation and Application of MSC-CM

During the surgery, MSC-CM was dissolved in 5 mL of saline. Pure porous β-TCP (Osferion; Olympus Terumo Biomaterials, Tokyo, Japan) was used as a scaffold in all cases. One gram of β-TCP was soaked in 3 mL of MSC-CM solution for at least 5 minutes and then used for grafting.

Surgical Procedures

All surgical procedures were conducted under local anesthesia. The SFE procedure was performed using the lateral window approach.17 Briefly, a window was created with a round diamond bur at the lateral maxillary sinus wall. After removal of the bone, the sinus membrane was elevated. The space created by this procedure was filled with the mixture of MSC-CM and β-TCP (MSC-CM group) or saline and β-TCP (β-TCP group). The lateral window was then covered with a titanium mesh (Jeil Ti mesh, Proceed, Tokyo, Japan). Implants were placed if the initial stability of fixtures was confirmed when SFE was performed. If the initial stability of any implant seemed inadequate, or if the residual alveolar bone was less than about 2 mm, only bone augmentation (without implant placement) was performed. Initial stability was confirmed by low mobility of fixtures upon implant placement. The mucoperiosteal flap was repositioned and sutured in the usual manner. A biopsy of the regenerated bone was performed during second-stage implant surgery or implant placement, which was scheduled 6 months after SFE (Table 1). All surgeries were performed by the same surgeon (W.K.).

Table 1
Table 1:
The Schedule of This Clinical Study

Clinical and Radiographic Observations

Before surgery, the safety of MSC-CM was evaluated. Briefly, the drug lymphocyte stimulation test and the patch test were performed to identify allergies to MSC-CM. Blood tests were also performed to check for organ dysfunction and inflammatory and allergic reactions before and after surgery. Computed tomography (CT) were performed before and after surgery. Hounsfield units (HU) of 3 randomly selected points from the augmented area 6 months after SFE were calculated using SIMPLANT 16 Pro software (Dentsply-Sankin K.K., Tokyo, Japan). The protocol for clinical evaluations in this study is shown in Table 1.

Histological Evaluation

Specimens were obtained from regions of newly formed bone 6 months after SFE using a trephine bar with an internal diameter of 2 mm. Explants were immersed in 10% formaldehyde, decalcified with K-CX solution (Falma Co., Tokyo, Japan), dehydrated with graded ethanol, cleared with xylene, and embedded in paraffin. Histological sections (5 μm thick) were created and stained with hematoxylin and eosin. The specimens were examined by light microscopy. Mature alveolar bone was excluded from the region of interest to avoid inclusion of the residual alveolar crest. Each specimen was divided into three parts (alveolar side, center, and sinus side), and the newly formed bone area was quantified and averaged from at least 3 random fields in each part using Adobe Photoshop Elements 13 (Adobe Systems Incorporated, Tokyo, Japan).

Statistical Analysis

All experiments were conducted in triplicate and repeated at least twice. Group mean values and standard deviations were calculated for each measured parameter. Statistical differences were evaluated with Welch's t test or Student's t test. A value of P < 0.05 was considered statistically significant.


Patients and Clinical Observations

Four patients (male:female = 2:2; mean age: 59.8 years) were enrolled in the MSC-CM group and two patients (male:female = 0:2; mean age: 70.0 years) were enrolled in the β-TCP group in this study. No systemic or local complications were reported during the study, and all patch tests, drug lymphocyte stimulation tests, and blood tests were negative. Implant placement was performed simultaneously with SFE except in the patients in the β-TCP group, who had thin alveolar bone (<2 mm) that reduced the initial stability of the implants. All implants were placed without any problems and showed good initial stability.

Radiographic Observations

CT images showed that the β-TCP scaffolds became indistinct around 6 months after implantation in both groups; however, the scaffolds seemed to be less prominent in the MSC-CM group. Abnormal bone resorption or notable edematous swelling of the maxillary sinus membrane was not observed in any case during the study. There was no statistical difference in HU between the two groups (Fig. 1).

Fig. 1
Fig. 1:
Radiographic observations. CT images of bone specimens 6 months after SFE. There was no statistical difference in HU between the MSC-CM and β-TCP groups.

Histological Observations

Newly formed bone was observed in each specimen, and remnants of β-TCP were resolved from the edge. Newly formed lamellar bone was observed only 6 months after SFE in the MSC-CM group (Figs. 2, A and 3, A). Many osteoblasts and osteoclasts were observed around the new bone and the β-TCP remnants in the MSC-CM group than in the β-TCP group (Figs. 2, B–D and 3, B–D). Furthermore, more vascularization was seen in the MSC-CM group than in the β-TCP group, especially at the center of the augmented area (Figs. 2, C and 3, C). Less replacement of β-TCP with newly formed bone was observed in the β-TCP group than in the MSC-CM group. In addition, newly formed bone in the β-TCP group consisted mainly of woven bone, and infiltration of inflammatory cells was observed (Fig. 3, B–D).

Fig. 2
Fig. 2:
Histological findings in the MSC-CM group.A, Outline of the specimens (×12.5). B, Alveolar side of the specimens (×100). C, Center of the specimens (×100). D, Sinus side of the specimens (×100). Newly formed lamellar bone and osteoblasts were seen throughout the specimens, and small amounts of residual β-TCP granules were seen only at the sinus floor of the augmented area, with some osteoclasts (BD). Furthermore, vascularization at the center of the augmented area was observed (C). Arrow: osteoblasts, Dotted arrow: osteoclasts. AB indicates alveolar bone; NB, newly formed bone; TCP, β-TCP granules; V, blood vessel.
Fig. 3
Fig. 3:
Histological findings in the β-TCP group.A, Outline of the specimens (×12.5). B, Alveolar side of the specimens (×100). C, Center of the specimens (×100). D, Sinus side of the specimens (×100). More infiltration of inflammatory cells (B) and β-TCP granules (C and D) were observed in the β-TCP group than in the MSC-CM group, and woven bone was prominent in the augmented area (C and D). Arrow: osteoblasts, Dotted arrow: osteoclasts, Arrow head: inflammatory cells. AB indicates alveolar bone; NB, newly formed bone; TCP, β-TCP granules; V, blood vessel.

The newly formed bone area was significantly greater in the MSC-CM group than in the β-TCP group (Fig. 4, A). Detailed assessments of the three parts of each specimen revealed that bone regeneration at the center of the augmented area was significantly greater in the MSC-CM group than in the β-TCP group (Fig. 4, B).

Fig. 4
Fig. 4:
Histological evaluations.A, Evaluation of total specimens. The newly formed bone area in the MSC-CM group was significantly larger than that in the β-TCP group (*P < 0.05). B, Evaluation of the 3 parts of the specimens. Each specimen was divided into the alveolar side, the center, and the sinus side, and the newly formed bone area in each part was evaluated. There was a significant difference between the 2 groups for the center of the specimens (*P < 0.05).


In this preliminary clinical study, we evaluated the effects of MSC-CM on bone regeneration after maxillary SFE. Our previous studies revealed that MSC-CM contains several cytokines, such as IGF-1, VEGF, and TGF-β1. IGF-1 regulates the migration of osteoblasts18 and MSCs,19 and sustained systemic or local infusion of IGF-1 enhances bone formation.20 VEGF is the main regulator of angiogenesis and also enhances survival and differentiation of endothelial cells that promote osteogenesis. TGF-β1 enhances the migration of osteoprogenitor cells and regulates cellular proliferation, differentiation, and extracellular matrix production.21 These growth factors act synergistically to promote vascularization, cell migration, osteogenesis, and bone regeneration.22–24

Our recent studies revealed that MSC-CM promotes earlier formation of new bone by enhancing endogenous stem cell migration, osteogenic differentiation, and angiogenesis in vitro and in vivo.12,13,15 We also reported that MSC-CM promoted early bone regeneration in a rabbit model of maxillary SFE.16 Cell proliferation and vascularization were increased 2 weeks after implantation of grafts impregnated with MSC-CM compared with controls, indicating that MSC-CM is effective in the early phase of bone regeneration and angiogenesis in the rabbit model. This suggests that in this study, early vascularization caused enhanced proliferation and migration of osteoprogenitor cells into the center of the augmented area, where the blood supply is more limited.

In this study, bone biopsies were performed about 6 months after SFE. The selection of this time period was based on the results of our preclinical and clinical studies as well as previous studies using PRP, because it was assumed that PRP and MSC-CM would have similar effects on new bone formation and bone substitute degradation.

PRP includes several osteoinductive growth factors, such as TGF-β, IGF-1, VEGF, PDGF, and epithelial growth factor.25 Although numerous studies of the effect of PRP on bone augmentation have been performed, evidence for the benefit of using PRP in bone augmentation procedures is equivocal and inconclusive.26 Several different study designs, outcome evaluations, and surgical techniques have been used in these studies, making it difficult to determine the benefit of using PRP. Moreover, the qualities of PRP vary. Individual patient factors strongly influence the characteristics of PRP,27 and PRP with leukocytes was reported to contain more TGF-β1 and PDGF than PRP without leukocytes.28,29 Furthermore, the secretion of growth factors from PRP begins within 10 minutes after coagulation; therefore, handling of PRP during surgery must be rigorous.

BMP-2 has been used widely with good efficacy in maxillary SFE. In general, the effects of this growth factor on bone regeneration are dose dependent. Therefore, high doses of BMP-2 are required to achieve sufficient bone volume.30 However, adverse events such as facial edema due to a localized inflammatory response have been caused by high, supraphysiological doses of BMP-2.3–5 INFUSE Bone Graft, which consists of recombinant human BMP-2 (1.5 mg/mL) applied to a bovine collagen carrier, is already on the market. The MSC-CM used in this study contained IGF-1, VEGF, and TGF-β1 at concentrations of 1386 ± 465, 468.5 ± 109, and 339.8 ± 14.4 pg/mL, respectively. In other words, MSC-CM contains a cocktail of growth factors at concentrations only 1/1000 of the concentration of BMP-2 in INFUSE. The low concentrations of multiple growth factors in MSC-CM could promote bone regeneration without triggering a severe inflammatory response.

Certain low-dose combinations of growth factors from MSC-CM may have potential for clinical use. In this study, CTs were performed to monitor the healing of the augmented area and detect adverse events such as abnormal bone resorption or sinusitis. Analysis of the CT images showed that there was no statistically significant difference in HU between the MSC-CM and β-TCP groups; however, histological analysis showed that there was more bone formation in the augmented area in the MSC-CM group than in the β-TCP group. Furthermore, the newly formed bone in the MSC-CM group, only 6 months after SFE, was mature lamellar bone. This indicates that MSC-CM can shorten the time needed for degradation and replacement of β-TCP and, therefore, the entire treatment time for SFE and implant-supported prosthetic rehabilitation of the atrophic edentulous maxilla. Because the main objective of this preliminary clinical study was to assess the safety of MSC-CM, the number of patients was small and we were unable to elucidate details of the effects of MSC-CM on bone regeneration. Further clinical study with a larger group of patients will be planned now.


The secretome of bone marrow–derived MSCs (MSC-CM) enhanced vascularization and early bone formation in maxillary SFE. This result suggests that this promising, novel therapeutic agent has the potential to improve bone quality in a shortened treatment time, thus accelerating implant-supported prosthetic rehabilitation of the atrophic edentulous maxilla.


The authors declare that they have no financial interest, either direct or indirect, in the products or information described in the article.


The ethics committee of Nagoya University Hospital approved this research protocol (No. 3437).

Roles/Contributions by Authors

W. Katagiri, J. Watanabe, and N. Toyama: Performed most of the experiments. W. Katagiri: Designed this study. J. Watanabe, N. Toyama, and K. Sakaguchi: Conducted the animal study. J. Watanabe, and M. Osugi: Performed the statistical analysis. W. Katagiri: Wrote the manuscript. H. Hibi: Wrote the critical review on the experimental process. All authors read and approved the final manuscript.


The authors thank members of the Department of Oral and Maxillofacial Surgery, Nagoya University Graduate School of Medicine, for their assistance. This work was supported in part by Grants-in-Aid for Scientific Research (B) from the Ministry of Health, Labour and Welfare of Japan (grant number 23592883).


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secretome; cytokine; paracrine; vasculization

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