Publications by authors named "Yoshinori Sumita"

43 Publications

Clinical Safety Assessment of Autologous Freeze-Drying Platelet-Rich Plasma for Bone Regeneration in Maxillary Sinus Floor Augmentation: A Pilot Study.

J Clin Med 2021 Apr 14;10(8). Epub 2021 Apr 14.

Department of Regenerative Oral Surgery, Institute of Biomedical Sciences, Nagasaki University Graduate School of Biomedical Sciences, 1-7-1 Sakamoto, Nagasaki 852-8588, Japan.

The purpose of this clinical study is to evaluate the safety and preliminary efficacy of autologous freeze-drying platelet-rich plasma (FD-PRP) on bone regeneration in maxillary sinus floor augmentation as a preliminary pilot study. Five patients that required sinus floor augmentation to facilitate the placement of dental implants participated in this clinical study. The PRP was prepared from the autologous peripheral blood and was lyophilized and stored at -20 °C for 4 weeks before surgery. At surgery, triple-concentrated FD-PRP (x3FD-PRP) mixed with synthetic bone grafting materials was rehydrated following the transplantation into the sinus floor. The primary outcome was a safety verification of x3FD-PRP, evaluated in terms of the clinical course and consecutive blood tests. The secondary outcome was clinical efficacy focused on bone regeneration in sinus floor augmentation evaluated by radiographic examination and implant stability. There were no adverse events, such as systemic complications, excessive inflammatory reactions, severe infection, or local site healing complications, besides those on the usual course associated with surgery. Vertical augmented height was maintained, and the initial stability of implants was achieved post-operatively in 6 months. The results obtained in this study suggest that x3FD-PRP can be used safely for bone engineering in clinical practice. Further studies are required to draw a conclusion concerning the efficacy of x3FD-PRP since this was a pilot study with a single arm and a small sample size.
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http://dx.doi.org/10.3390/jcm10081678DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8070716PMC
April 2021

Gene-activated matrix harboring a miR20a-expressing plasmid promotes rat cranial bone augmentation.

Regen Biomater 2021 Mar 13;8(2):rbaa060. Epub 2021 Mar 13.

Department of Regenerative Oral Surgery, Unit of Translational Medicine, Nagasaki University Graduate School of Biomedical Sciences, 1-7-1 Sakamoto, Nagasaki 852-8588, Japan.

Gene-activated matrix (GAM) has a potential usefulness in bone engineering as an alternate strategy for the lasting release of osteogenic proteins but efficient methods to generate non-viral GAM remain to be established. In this study, we investigated whether an atelocollagen-based GAM containing naked-plasmid () DNAs encoding microRNA (miR) 20a, which may promote osteogenesis via multiple pathways associated with the osteogenic differentiation of mesenchymal stem/progenitor cells (MSCs), facilitates rat cranial bone augmentation. First, we confirmed the osteoblastic differentiation functions of generated DNA encoding miR20a (miR20a) , and its transfection regulated the expression of several of target genes, such as Bambi1 and PPARγ, in rat bone marrow MSCs and induced the increased expression of BMP4. Then, when GAMs fabricated by mixing 100 μl of 2% bovine atelocollagen, 20 mg β-TCP granules and 0.5 mg (3.3 μg/μl) AcGFP plasmid-vectors encoding miR20a were transplanted to rat cranial bone surface, the promoted vertical bone augmentation was clearly recognized up to 8 weeks after transplantation, as were upregulation of VEGFs and BMP4 expressions at the early stages of transplantation. Thus, GAM-based miR delivery may provide an alternative non-viral approach by improving transgene efficacy via a small sequence that can regulate the multiple pathways.
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http://dx.doi.org/10.1093/rb/rbaa060DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7955717PMC
March 2021

Resident CD34-positive cells contribute to peri-endothelial cells and vascular morphogenesis in salivary gland after irradiation.

J Neural Transm (Vienna) 2020 11 6;127(11):1467-1479. Epub 2020 Oct 6.

Institute of Anatomy and Cell Biology, University of Würzburg, Würzburg, Germany.

Salivary gland (SG) hypofunction is a common post-radiotherapy complication. Besides the parenchymal damage after irradiation (IR), there are also effects on mesenchymal stem cells (MSCs) which were shown to contribute to regeneration and repair of damaged tissues by differentiating into stromal cell types or releasing vesicles and soluble factors supporting the healing processes. However, there are no adequate reports about their roles during SG damage and regeneration so far. Using an irradiated SG mouse model, we performed certain immunostainings on tissue sections of submandibular glands at different time points after IR. Immunostaining for CD31 revealed that already one day after IR, vascular impairment was induced at the level of capillaries. In addition, the expression of CD44-a marker of acinar cells-diminished gradually after IR and, by 20 weeks, almost disappeared. In contrast, the number of CD34-positive cells significantly increased 4 weeks after IR and some of the CD34-positive cells were found to reside within the adventitia of arteries and veins. Laser confocal microscopic analyses revealed an accumulation of CD34-positive cells within the area of damaged capillaries where they were in close contact to the CD31-positive endothelial cells. At 4 weeks after IR, a fraction of the CD34-positive cells underwent differentiation into α-SMA-positive cells, which suggests that they may contribute to regeneration of smooth muscle cells and/or pericytes covering the small vessels from the outside. In conclusion, SG-resident CD34-positive cells represent a population of progenitors that could contribute to new vessel formation and/or remodeling of the pre-existing vessels after IR and thus, might be an important player during SG tissue healing.
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http://dx.doi.org/10.1007/s00702-020-02256-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7578140PMC
November 2020

Phase 1 clinical study of cell therapy with effective-mononuclear cells (E-MNC) for radiogenic xerostomia (first-in-human study) (FIH study on E-MNC therapy for radiogenic xerostomia).

Medicine (Baltimore) 2020 Jun;99(26):e20788

Department of Regenerative Oral Surgery, Unit of Translational Medicine.

Background: Treatment for most patients with head and neck cancers includes ionizing radiation with or without chemotherapy. This treatment causes irreversible damage to salivary glands in the irradiation field accompanied by a loss of fluid-secreting acinar cells and a considerable decrease of saliva secretion. There is currently no adequate conventional treatment for this condition. In recent years, we developed an effective culture method to enhance the anti-inflammatory and vasculogenic phenotypes of peripheral blood mononuclear cells (PBMNCs), and such effectively conditioned PBMNC (E-MNC) therapy has shown promising improvements to the function of radiation-injured salivary glands in preclinical studies. However, the safety and effect of E-NMC therapy have yet assessed in human. The objective of this ongoing first-in-man study is to assess the safety, tolerability, and in part the efficacy of E-MNC therapy for treating radiation-induced xerostomia.

Methods/design: This phase 1 first-in-man study is an open-label, single-center, two-step dose escalation study. A total of 6 patients, who had no recurrence of head and neck cancer over 5 years following radiation therapy and suffered from radiation-induced xerostomia, will receive a transplantation of E-NMCs derived from autologous PBMNCs to a submandibular gland. The duration of the intervention will be 1 year. To analyze the recovery of salivary secretion, a gum test will be performed. To analyze the recovery of atrophic salivary glands, computed tomography (CT), and magnetic resonance imaging (MRI) of salivary glands will be conducted. The primary endpoint is the safety of the protocol. The secondary endpoints are the changes from baseline in whole saliva secretion and salivary gland atrophy.

Discussion: This will be the first clinical study of regenerative therapy using E-MNCs for patients with severe radiation-induced xerostomia. The results of this study are expected to contribute to developing the low-invasive cell-based therapy for radiation-induced xerostomia.

Trial Registration: This study was registered with the Japan Registry of Clinical Trials (http://jrct.niph.go.jp) as jRCTb070190057.
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http://dx.doi.org/10.1097/MD.0000000000020788DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7328916PMC
June 2020

Do not keep it simple: recent advances in the generation of complex organoids.

J Neural Transm (Vienna) 2020 11 8;127(11):1569-1577. Epub 2020 May 8.

Institute of Anatomy and Cell Biology, University of Würzburg, Würzburg, Germany.

3D cell culture models which closely resemble real human tissues are of high interest for disease modelling, drug screening as well as a deeper understanding of human developmental biology. Such structures are termed organoids. Within the last years, several human organoid models were described. These are usually stem cell derived, arise by self-organization, mimic mechanisms of normal tissue development, show typical organ morphogenesis and recapitulate at least some organ specific functions. Many tissues have been reproduced in vitro such as gut, liver, lung, kidney and brain. The resulting entities can be either derived from an adult stem cell population, or generated from pluripotent stem cells using a specific differentiation protocol. However, many organoid models only recapitulate the organs parenchyma but are devoid of stromal components such as blood vessels, connective tissue and inflammatory cells. Recent studies show that the incorporation of endothelial and mesenchymal cells into organoids improved their maturation and might be required to create fully functional micro-tissues, which will allow deeper insights into human embryogenesis as well as disease development and progression. In this review article, we will summarize and discuss recent works trying to incorporate stromal components into organoids, with a special focus on neural organoid models.
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http://dx.doi.org/10.1007/s00702-020-02198-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7577912PMC
November 2020

A stable protocol for the fabrication of transplantable human oral mucosal epithelial cell sheets for clinical application.

Regen Ther 2020 Jun 16;14:87-94. Epub 2020 Jan 16.

Institute of Advanced Biomedical Engineering and Science, Tokyo Women's Medical University (TWIns), 8-1 Kawada-Cho, Shinjuku-ku, Tokyo, 162-8666, Japan.

Introduction: Cultured stratified epithelial cell sheets have been clinically utilized as transplantable grafts for the regeneration of epithelial tissues, such as the esophagus, cornea, skin, and intraoral cavity. These cell sheets are expected to gain widespread use as regenerative medicine products and save many patients. For this purpose, establishing and disseminating the stale protocol of fabricating the cell sheet is crucial. The fabrication of cultured stratified epithelial cell sheets consists of many important steps, and since the patients' epithelial cell conditions vary widely and are sometimes unstable, the qualities of the epithelial cell grafts are likewise potentially unstable. Therefore, in this paper, we report the stable protocol for fabrication of the transplantable cell sheet particularly from patient-derived oral mucosal tissues.

Methods: Serum extracted from blood and buccal mucosal tissue were collected in Nagasaki University and transported to Tokyo Women's Medical University. Oral mucosal epithelial cells were collected by minimum trypsin method, and this treatment was studied whether to be a critical procedure. After 14 days cultivation, cultured cells were examined whether to be transplantable as cell sheets.

Results: We successfully transported buccal mucosal tissue and serum without damage and contamination. Oral mucosal epithelial cells were collected with high viability by minimum trypsin method. Finally, we succeeded to stably fabricate oral mucosal epithelial cell sheets in all 10 patients.

Conclusions: We established a stable protocol for the fabrication of human oral mucosal epithelial cell sheets and their transportation in clinical settings in this study. These methodologies could also be basis for transplantation therapy using cultured cell sheets of various types other than oral mucosal epithelial cell and will contribute largely to the future development of regenerative medicine.
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http://dx.doi.org/10.1016/j.reth.2019.11.007DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6970131PMC
June 2020

Intra-Bone Marrow Administration of Mesenchymal Stem/Stromal Cells Is a Promising Approach for Treating Osteoporosis.

Stem Cells Int 2019 12;2019:4214281. Epub 2019 Nov 12.

Department of Regenerative Oral Surgery, Unit of Translational Medicine, Nagasaki University Graduate School of Biomedical Sciences, Nagasaki, Japan.

Mesenchymal stem/stromal cells (MSCs) are known to be useful for treating local bone diseases. However, it is not known if MSCs are effective for treating systemic bone diseases, as the risk for mortality following intravenous MSC administration has hindered research progress. In this study, we compared the safety and efficacy of intra-bone marrow and intravenous administration of MSCs for the treatment of ovariectomy- (OVX-) induced osteoporosis. Cells capable of forming bone were isolated from the murine compact bones and expanded in culture. Relatively pure MSCs possessing increased potential for cell proliferation, osteogenic differentiation, and inhibition of osteoclastogenesis were obtained by magnetic-activated cell sorting with the anti-Sca-1 antibody. Sca-1-sorted MSCs were administered to OVX mice, which were sacrificed 1 month later. We observed that 22% of the mice died after intravenous administration, whereas none of the mice died after intra-bone marrow administration. With respect to efficacy, intravenous administration improved bone mineral density (BMD) by increasing bone mineral content without affecting bone thickness, whereas intra-bone marrow administration improved BMD by increasing both bone mineral content and bone thickness. These results indicate that intra-bone marrow administration of pure MSCs is a safer and more effective approach for treating osteoporosis.
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http://dx.doi.org/10.1155/2019/4214281DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6875206PMC
November 2019

Anti-inflammatory and vasculogenic conditioning of peripheral blood mononuclear cells reinforces their therapeutic potential for radiation-injured salivary glands.

Stem Cell Res Ther 2019 10 17;10(1):304. Epub 2019 Oct 17.

Department of Regenerative Oral Surgery, Unit of Translational Medicine, Nagasaki University Graduate School of Biomedical Sciences, Nagasaki, Japan.

Background: There are currently no effective treatments available for patients with irreversible loss of salivary gland (SG) function caused by radiation therapy for head and neck cancer. In this study, we have developed an effective culture method to enhance the anti-inflammatory and vasculogenic phenotypes of peripheral blood mononuclear cells (PBMNCs) and investigated whether such effectively conditioned PBMNCs (E-MNCs) could regenerate radiation-injured SGs and ameliorate salivary secretory function in mice.

Methods: Mouse PBMNCs were expanded in primary serum-free culture with five vasculogenic proteins for 5 days, and then the resulting cells (E-MNCs) were analyzed for their characteristics. Subsequently, 5 × 10 E-MNCs (labeled with EGFP in some experiments) were injected intra-glandularly into a mouse model of radiation-injured atrophic submandibular glands. After 2-3 weeks, the submandibular glands were harvested, and then the injected E-MNCs were tracked. Four, 8, and 12 weeks after irradiation (IR), salivary outputs were measured to evaluate the recovery of secretory function, and the gland tissues were harvested for histological and gene expression analyses to clarify the effects of cell transplantation.

Results: The resulting E-MNCs contained an enriched population of definitive CD11b/CD206-positive (M2 macrophage-like) cells and showed anti-inflammatory and vasculogenic characteristics. Salivary secretory function in E-MNC-transplanted mice gradually recovered after 4 weeks post-irradiation (post-IR) and reached 3.8-fold higher than that of non-transplanted mice at 12 weeks. EGFP-expressing E-MNCs were detected in a portion of the vascular endothelium and perivascular gland tissues at 2 weeks post-IR, but mainly in some microvessels at 3 weeks. Between 4 and 12 weeks post-IR, mRNA expression and histological analyses revealed that E-MNC transplantation reduced the expression of inflammatory genes and increased the level of tissue-regenerative activities such as stem cell markers, cell proliferation, and blood vessel formation. At 12 weeks post-IR, the areas of acinar and ductal cells regenerated, and the glands had less fibrosis.

Conclusions: This effective conditioning of PBMNCs is a simple, rapid, and efficient method that provides a non-invasive source of therapeutic cells for regenerating radiation-injured atrophic SGs.
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http://dx.doi.org/10.1186/s13287-019-1414-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6798785PMC
October 2019

Regenerative dentistry in periodontics.

Saudi Dent J 2019 Jul 13;31(3):301-302. Epub 2019 May 13.

Division of Dental Anesthesiology, Niigata University Graduate School of Medical and Dental Sciences, Niigata, Japan.

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http://dx.doi.org/10.1016/j.sdentj.2019.05.002DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6626286PMC
July 2019

Systemic administration of quality- and quantity-controlled PBMNCs reduces bisphosphonate-related osteonecrosis of jaw-like lesions in mice.

Stem Cell Res Ther 2019 07 16;10(1):209. Epub 2019 Jul 16.

Department of Applied Prosthodontics, Institute of Biomedical Sciences, Nagasaki University, 1-7-1, Sakamoto, Nagasaki, 852-8588, Japan.

Background: Definitive treatment strategies for bisphosphonate-related osteonecrosis of the jaw (BRONJ) have not been developed. Cell-based therapy is an attractive treatment method for intractable diseases in the medical and dental fields; however, approval has been challenging in dentistry. Recently, we developed quality- and quantity (QQ)-controlled peripheral blood mononuclear cells (PBMNCs) that have anti-inflammatory and pro-angiogenesis effects. The aim of this study was to investigate the effects of QQ-controlled PBMNC transplantation on BRONJ-like lesions in mice.

Methods: To create high-prevalence BRONJ-like lesions, cyclophosphamide (CY) and zoledronate (ZA) were used with tooth extraction. Drug treatment was performed for 5 weeks. QQ-controlled PBMNC transplantation was performed immediately following tooth extraction of both maxillary first molars at 3 weeks after drug administration. Mice were euthanized at 2 weeks post-extraction. Histomorphometric and immunohistochemical analyses, microcomputed tomography assessment, and quantitative polymerase chain reaction evaluation were conducted using maxillae and long bones.

Results: ZA effects on long bones were noted, regardless of CY. Severely inhibited osseous and soft tissue wound healing of tooth extraction sockets was induced by CY/ZA combination therapy, which was diagnosed as BRONJ-like lesions. QQ-controlled PBMNC transplantation reduced BRONJ-like lesions by improving soft tissue healing with increased M1 and M2 macrophages and enhanced neovascularization in the connective tissue of tooth extraction sockets. QQ-controlled PBMNC transplantation also reduced inflammation by decreasing polymorphonuclear cells and TNF-α expression in the tooth extraction sockets. Additionally, QQ-controlled PBMNC transplantation partially improved osseous healing of tooth extraction sockets. Interestingly, only 20,000 QQ-controlled PBMNCs per mouse induced these transplantation effects. QQ-controlled PBMNC transplantation did not affect the systemic microenvironment.

Conclusions: Our findings suggest that transplantation of a small amount of QQ-controlled PBMNCs may become novel therapeutic or prevention strategies for BRONJ without any adverse side effects.
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http://dx.doi.org/10.1186/s13287-019-1308-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6636115PMC
July 2019

First clinical application of octacalcium phosphate collagen composite on bone regeneration in maxillary sinus floor augmentation: A prospective, single-arm, open-label clinical trial.

J Biomed Mater Res B Appl Biomater 2020 01 13;108(1):243-252. Epub 2019 Apr 13.

Department of Regenerative Oral Surgery, Nagasaki University Graduate School of Biomedical Sciences, Nagasaki, Japan.

The overall objective of this study was to assess the safety and efficacy of OCP/Col as a bone substitute material for bone regeneration during sinus floor augmentation. Maxillary sinus floor augmentation was performed thorough lateral window approach. According to the height of host bone, simultaneous approach (≥5 mm) or staged approach (less than 5 mm) was applied. In this research, clinical findings of dental implant treatment after setting the restorations were set as a primary endpoint in both approaches (infection, inflammation around the implant, movement of the implant, pain, sensory disorder, and bone resorption around the implant body on radiological evaluation.). In staged approach, histological evaluation of bone biopsy specimen was also conducted. As secondary endpoints, hounsfield unit (HU) value, vertical bone height, implant stability quotient (ISQ), and adverse events during the research were evaluated. In all cases, as a primary endpoint, clinical findings after setting the restorations were uneventful with no adverse events. Histological structure demonstrated mature bone derived from OCP/Col. In the ossified area, osteogenesis was observed around OCP granules, and osteoblast-like cells were arrayed around OCP granules. Osteocyte encapsulation was recognized in the new bone. HU increased over time with both approaches. Vertical bone height significantly increased at 3 months postoperatively, and maintained during follow-up. ISQ increased with both approaches. In particular, ISQ was significantly increased with the staged approach. This clinical trial demonstrated the safety and efficacy of OCP/Col for bone regeneration in maxillary sinus floor augmentation. © 2019 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater 108B:243-252, 2020.
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http://dx.doi.org/10.1002/jbm.b.34384DOI Listing
January 2020

Alveolar bone preservation by a hydroxyapatite/collagen composite material after tooth extraction.

Clin Oral Investig 2019 May 9;23(5):2413-2419. Epub 2018 Oct 9.

Department of Regenerative Oral Surgery, Institute of Biomedical Sciences, Nagasaki University, 1-7-1 Sakamoto, Nagasaki, 852-8588, Japan.

Objective: The aim of this study was to assess the effectiveness of a hydroxyapatite/collagen composite material (HAp/Col) for preservation of alveolar bone after tooth extraction.

Materials And Methods: HAp/Col was applied to the alveolus bone ridge preservation after tooth extraction, because of subsequent dental implant placement in 35 regions of 24 patients (mean age, 59.3 years; range, 25-81 years). Cone beam computed tomography was used to assess changes in alveolar bone at the extraction site before and at 3 months (mean, 13.7 weeks; range, 10-17 weeks) after tooth extraction. Changes in height and width of the alveolar bone were measured to evaluate bone reduction after surgery. Bone biopsy was performed at 11 regions of dental implant placement to observe bone regeneration and remaining material in the extraction socket.

Results: The alveolar bone height was decreased by 0.00 ± 2.44 mm at the buccal side and 0.35 ± 1.73 mm at the lingual side, while the width was decreased by 1.02 ± 1.64 mm at 3 months after surgery. The middle of the socket floor was elevated by 5.71 ± 3.45 mm at 3 months after surgery. Bone biopsy specimens revealed no remaining implanted material, and approximately 49.79 ± 14.41% of the specimens were occupied by bone tissue.

Conclusions: According to the result of this study, HAp/Col is a reliable material to presearve alveolar bone after tooth extraction.

Clinical Relevance: HAp/Col contributes dental implant treatment due to maintain the alveolar bone after tooth extraction.
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http://dx.doi.org/10.1007/s00784-018-2705-6DOI Listing
May 2019

Bone marrow concentrate promotes bone regeneration with a suboptimal-dose of rhBMP-2.

PLoS One 2018 18;13(1):e0191099. Epub 2018 Jan 18.

Department of Regenerative Oral Surgery, Unit of Translational Medicine, Nagasaki University Graduate School of Biomedical Sciences, Nagasaki, Japan.

Bone marrow concentrate (BMC), which is enriched in mononuclear cells (MNCs) and platelets, has recently attracted the attention of clinicians as a new optional means for bone engineering. We previously reported that the osteoinductive effect of bone morphogenetic protein-2 (BMP-2) could be enhanced synergistically by co-transplantation of peripheral blood (PB)-derived platelet-rich plasma (PRP). This study aims to investigate whether BMC can effectively promote bone formation induced by low-dose BMP-2, thereby reducing the undesirable side-effects of BMP-2, compared to PRP. Human BMC was obtained from bone marrow aspirates using an automated blood separator. The BMC was then seeded onto β-TCP granules pre-adsorbed with a suboptimal-dose (minimum concentration to induce bone formation at 2 weeks in mice) of recombinant human (rh) BMP-2. These specimens were transplanted subcutaneously to the dorsal skin of immunodeficient-mice and the induction of ectopic bone formation was assessed 2 and 4 weeks post-transplantation. Transplantations of five other groups [PB, PRP, platelet-poor plasma (PPP), bone marrow aspirate (BM), and BM-PPP] were employed as experimental controls. Then, to clarify the effects on vertical bone augmentation, specimens from the six groups were transplanted for on-lay placement on the craniums of mice. The results indicated that BMC, which contained an approximately 2.5-fold increase in the number of MNCs compared to PRP, could accelerate ectopic bone formation until 2 weeks post-transplantation. On the cranium, the BMC group promoted bone augmentation with a suboptimal-dose of rhBMP-2 compared to other groups. Particularly in the BMC specimens harvested at 4 weeks, we observed newly formed bone surrounding the TCP granules at sites far from the calvarial bone. In conclusion, the addition of BMC could reduce the amount of rhBMP-2 by one-half via its synergistic effect on early-phase osteoinduction. We propose here that BMC transplantation facilitates the clinical use of rhBMP-2 as an alternative strategy for bone engineering.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0191099PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5773187PMC
February 2018

Optimal timing and frequency of bone marrow soup therapy for functional restoration of salivary glands injured by single-dose or fractionated irradiation.

J Tissue Eng Regen Med 2018 02 8;12(2):e1195-e1205. Epub 2017 Nov 8.

Craniofacial Tissue Engineering and Stem Cells Laboratory, Faculty of Dentistry, McGill University, Montreal, Canada.

Injections of bone marrow (BM) cell extract, known as 'BM soup', were previously reported to mitigate ionizing radiation (IR) injury to salivary glands (SGs). However, the optimal starting time and frequency to maintain BM soup therapeutic efficacy remains unknown. This study tested the optimal starting time and frequency of BM soup injections in mice radiated with either a single dose or a fractionated dose. First, BM soup treatment was started at 1, 3 or 7 weeks post-IR; positive (non-IR) and negative (IR) control mice received injections of saline (vehicle control). Second, BM soup-treated mice received injections at different frequencies (1, 2, 3 and 5 weekly injections). Third, a 'fractionated-dose radiation' model to injure mouse SGs was developed (5 Gy × 5 days) and compared with the single high dose radiation model. All mice (n = 65) were followed for 16 weeks post-IR. The results showed that starting injections of BM soup between 1 and 3 weeks mitigated the effect of IR-induced injury to SGs and improved the restoration of salivary function. Although the therapeutic effect of BM soup lessens after 8 weeks, it can be sustained by increasing the frequency of weekly injections. Moreover, both single-dose and fractionated-dose radiation models are efficient and comparable in inducing SG injury and BM soup treatments are effective in restoring salivary function in both radiation models. In conclusion, starting injections of BM soup within 3 weeks post-radiation, with 5 weekly injections, maintains 90-100% of saliva flow in radiated mice.
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http://dx.doi.org/10.1002/term.2513DOI Listing
February 2018

Soft tissue engineering with micronized-gingival connective tissues.

J Cell Physiol 2018 Jan 3;233(1):249-258. Epub 2017 May 3.

Department of Regenerative Oral Surgery, Nagasaki University Graduate School of Biomedical Sciences, Nagasaki, Japan.

The free gingival graft (FGG) and connective tissue graft (CTG) are currently considered to be the gold standards for keratinized gingival tissue reconstruction and augmentation. However, these procedures have some disadvantages in harvesting large grafts, such as donor-site morbidity as well as insufficient gingival width and thickness at the recipient site post-treatment. To solve these problems, we focused on an alternative strategy using micronized tissue transplantation (micro-graft). In this study, we first investigated whether transplantation of micronized gingival connective tissues (MGCTs) promotes skin wound healing. MGCTs (≤100 µm) were obtained by mincing a small piece (8 mm ) of porcine keratinized gingiva using the RIGENERA system. The MGCTs were then transplanted to a full skin defect (5 mm in diameter) on the dorsal surface of immunodeficient mice after seeding to an atelocollagen matrix. Transplantations of atelocollagen matrixes with and without micronized dermis were employed as experimental controls. The results indicated that MGCTs markedly promote the vascularization and epithelialization of the defect area 14 days after transplantation compared to the experimental controls. After 21 days, complete wound closure with low contraction was obtained only in the MGCT grafts. Tracking analysis of transplanted MGCTs revealed that some mesenchymal cells derived from MGCTs can survive during healing and may function to assist in wound healing. We propose here that micro-grafting with MGCTs represents an alternative strategy for keratinized tissue reconstruction that is characterized by low morbidity and ready availability.
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http://dx.doi.org/10.1002/jcp.25871DOI Listing
January 2018

A Simplified and Systematic Method to Isolate, Culture, and Characterize Multiple Types of Human Dental Stem Cells from a Single Tooth.

Methods Mol Biol 2017 ;1553:191-207

Craniofacial Tissue Engineering and Stem Cells Laboratory, Faculty of Dentistry, McGill University, 3640 University Street, M43, Montreal, QC, Canada.

This chapter describes a simplified method that allows the systematic isolation of multiple types of dental stem cells such as dental pulp stem cells (DPSC), periodontal ligament stem cells (PDLSC), and stem cells of the apical papilla (SCAP) from a single tooth. Of specific interest is the modified laboratory approach to harvest/retrieve the dental pulp tissue by minimizing trauma to DPSC by continuous irrigation, reduction of frictional heat from the bur rotation, and reduction of the bur contact time with the dentin. Also, the use of a chisel and a mallet will maximize the number of live DPSC for culture. Steps demonstrating the potential for multiple cell differentiation lineages of each type of dental stem cell into either osteocytes, adipocytes, or chondrocytes are described. Flow cytometry, with a detailed strategy for cell gating and analysis, is described to verify characteristic markers of human mesenchymal multipotent stromal cells (MSC) from DPSC, PDLSC, or SCAP for subsequent experiments in cell therapy and in tissue engineering. Overall, this method can be adapted to any laboratory with a general setup for cell culture experiments.
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http://dx.doi.org/10.1007/978-1-4939-6756-8_15DOI Listing
February 2018

Efficacy of freeze-dried platelet-rich plasma in bone engineering.

Arch Oral Biol 2017 Jan 15;73:172-178. Epub 2016 Oct 15.

Department of Regenerative Oral Surgery, Unit of Translational Medicine, Nagasaki University Graduate School of Biomedical Sciences, 1-7-1 Sakamoto, Nagasaki-shi, Nagasaki 852-8102, Japan. Electronic address:

Objective: Platelet-rich plasma (PRP) is typically isolated and applied immediately after preparation, making it both a time- and labor-intensive addition to the operative procedure. Thus, it would be convenient if PRP could be preserved. We evaluated the efficacy of freeze-dried PRP (FD-PRP), as compared with freshly isolated PRP (f-PRP) for bone engineering.

Design: FD-PRP was prepared by lyophilization of f-PRP and was subsequently preserved at -20°C for one month. It was then rehydrated with an equal or 1/3 amount of distilled water (×1FD-PRP, ×3FD-PRP, respectively), and we assessed its gelation properties and the release of growth factors (PDGF-BB, TGF-β1, and VEGF). We also examined the bone forming ability with onlay-grafting on mice calvaria using β-TCP granules as a scaffold.

Results: FD-PRP showed comparable gelation as f-PRP. In terms of growth factor release,×1FD-PRP released identical concentrations of PDGF-BB and TGF-β1 to f-PRP, while ×3FD-PRP released approximately 3-fold concentrations when compared with f-PRP. In vivo, ×1FD-PRP promoted identical levels of the bone formation as f-PRP, and ×3FD-PRP induced more abundant bone formation.

Conclusions: These results suggest that f-PRP can be stored without functional loss by freeze-drying and the concentration of PRP may improve its efficacy in bone engineering.
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http://dx.doi.org/10.1016/j.archoralbio.2016.10.006DOI Listing
January 2017

Bone Regeneration Using Dentin Matrix Depends on the Degree of Demineralization and Particle Size.

PLoS One 2016 21;11(1):e0147235. Epub 2016 Jan 21.

Department of Regenerative Oral Surgery, Unit of Translational Medicine, Nagasaki University Graduate School of Biomedical Sciences, Nagasaki, Japan.

Objectives: This study aimed to examine the influence of particle size and extent of demineralization of dentin matrix on bone regeneration.

Materials And Methods: Extracted human teeth were pulverized and divided into 3 groups according to particle size; 200, 500, and 1000 μm. Each group was divided into 3 groups depending on the extent of demineralization; undemineralized dentin (UDD), partially demineralized dentin matrix (PDDM), and completely demineralized dentin matrix (CDDM). The dentin sample was implanted into rat calvarial bone defects. After 4 and 8 weeks, the bone regeneration was evaluated with micro-CT images, histomorphometric and immunohistochemical analyses. Osteoblasts were cultured on UDD and DDM to evaluate the cell attachment using electron microscope.

Results: Micro-CT images and histological observation revealed that CDDM had largely resorbed but UDD had not, and both of them induced little bone formation, whereas all particle sizes of PDDM induced more new bone, especially the 1000 μm. Electron microscopic observation showed osteoblasts attached to DDM but not to UDD.

Conclusions: PDDM with larger particle size induced prominent bone regeneration, probably because PDDM possessed a suitable surface for cell attachment. There might be an exquisite balance between its resorption and bone formation on it. PDDM could be considered as a potential bone substitute.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0147235PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4721666PMC
August 2016

Onlay bone augmentation on mouse calvarial bone using a hydroxyapatite/collagen composite material with total blood or platelet-rich plasma.

Arch Oral Biol 2016 Jan 22;61:23-7. Epub 2015 Oct 22.

Division of Dentistry and Oral Surgery, Department of Sensory and Locomotor Medicine, Faculty of Medical Sciences, University of Fukui, Japan.

Objective: The aim of this study was to assess newly formed onlay bone on mouse calvarial bone using a new artificial bone material, a hydroxyapatite/collagen composite, with total blood or platelet-rich plasma.

Design: The hydroxyapatite/collagen composite material with normal saline, total blood or platelet-rich plasma was transplanted on mouse calvarial bone. The mice were sacrificed and the specimens were harvested four weeks after surgery. The newly formed bone area was measured on hematoxylin and eosin stained specimens using Image J software.

Results: The hydroxyapatite/collagen composite materials with total blood or platelet-rich plasma induced a significantly greater amount of newly formed bone than that with normal saline. Moreover, bone marrow was observed four weeks after surgery in the transplanted materials with total blood or platelet-rich plasma but not with normal saline. However, there were no significant differences in the amount of newly formed bone between materials used with total blood versus platelet-rich plasma.

Conclusions: The hydroxyapatite/collagen composite material was valid for onlay bone augmentation and this material should be soaked in total blood or platelet-rich plasma prior to transplantation.
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http://dx.doi.org/10.1016/j.archoralbio.2015.10.012DOI Listing
January 2016

Transient Exposure to Hypoxic and Anoxic Oxygen Concentrations Promotes Either Osteogenic or Ligamentogenic Characteristics of PDL Cells.

Biores Open Access 2015 1;4(1):175-87. Epub 2015 Feb 1.

Department of Regenerative Oral Surgery, Unit of Translational Medicine, Graduate School of Biomedical Sciences, Nagasaki University , Nagasaki, Japan .

The periodontal ligament (PDL) has a reservoir of mesenchymal stem cells (MSCs) and this tissue is easily available following teeth removal procedures. However, PDL-derived cells (PDLCs) availability for tissue engineering is limited because they are heterogeneous cells at various differentiation and lineage commitments. Therefore, efficient culture conditions to increase MSCs number are needed to use PDLCs in tissue engineering. Recent reports indicate that low-oxygen conditions amplified stem/progenitor cell numbers and inhibited cell differentiation. Our aim was to establish which low-oxygen culture conditions favored bone or tendon/ligament regeneration in cultured PDLCs. Human PDLCs were cultured and exposed to either hypoxic (O2≤5%) or anoxic (O2<0.1%) oxygen conditions in low-glucose/serum-free media for 24 hours. After 24 h, as expected, cell survival was significantly less in PDLCs exposed to anoxic conditions as compared with cells under normal or hypoxic conditions. PDLCs exposed to hypoxic conditions had the highest percentages for MSC markers (CD105, CD166, Stro-1). For both hypoxic and anoxic conditions, stem cell marker genes (oct4, sox2, p75) were upregulated after 6 h. At 24 h, these stem cell markers were maintained in PDLCs under hypoxic condition. Interestingly under anoxic conditions, expression of scleraxis gene (a key transcription factor for tendo/ligamentogenesis) was upregulated markedly. When hypoxic PDLCs were subcultured into osteogenic medium, in vitro calcification and prominent in vivo bone formation in mice calvaria were observed. When anoxic PDLCs were subcultured into tendo/ligamentogenic medium, expression of aggrecan (a mature tenogenic gene) increased remarkably. No obvious differences were detectable on chondrogenic and adipogenic inducibilities. We propose that transient exposure to low-oxygen during the culture enhanced MSC population in PDL. In addition, different low-oxygen concentrations favored osteogenic or tendo/ligamentogenic inducibilities of cultured PDLCs.
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http://dx.doi.org/10.1089/biores.2014.0049DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4497711PMC
August 2015

Gene-Activated Matrix Comprised of Atelocollagen and Plasmid DNA Encoding BMP4 or Runx2 Promotes Rat Cranial Bone Augmentation.

Biores Open Access 2015 1;4(1):164-74. Epub 2015 Feb 1.

Department of Regenerative Oral Surgery, Unit of Translational Medicine, Graduate School of Biomedical Sciences, Nagasaki University , Nagasaki, Japan .

To date, therapeutic method for in vivo gene delivery has not been established on bone engineering though its potential usefulness has been suggested. For clinical applications, an effective condition should be developed to transfer the genes in vivo without any transfection reagents or virus vectors. In this study, to facilitate the clinical setting of this strategy, particularly aimed at atrophic bone repair, we simply investigated whether manufactured gene-activated matrix (GAM) with atelocollagen containing a certain amount of plasmid (p) DNA encoding osteogenic proteins could augment the cranial bone in rat. GAMs were manufactured by mixing 0.02, 0.1, or 1 mg of AcGFP plasmid vectors harboring cDNA of BMP4 (pBMP4) or Runx2 (pRunx2) with 2% bovine atelocollagen and β-tricalcium phosphate granules. Before manufacturing GAMs, to determine the biological activity of generated pDNAs, we confirmed GFP expression and increased level of alkaline phosphatase activities in MC3T3-E1 cells transfected with pBMP4 or pRunx2 during culture. Then, GAMs were lyophilized and transplanted to onlay placement on the cranium. At 2 weeks of transplantation, GFP-expressing cells could be detectable in only GAMs containing 1 mg of AcGFP plasmid vectors. Then, at 4 weeks, significant bone formation was recognized in GAMs containing 1 mg of pDNAs encoding BMP4 or Runx2 but not in 0.02 or 0.1 mg of GAMs. These newly formed bone tissues surrounded by osteocalcin-stained area were augmented markedly until 8 weeks after transplantation. In contrast, minimal bone formation was observed in GAMs without harboring cDNA of osteogenic proteins. Meanwhile, when GAMs were transplanted to the cranial bone defect, bone formation was detectable in specimens containing 1 mg of pBMP4 or pRunx2 at 8 weeks as well. Thus, atelocollagen-based GAM reliably could form the engineered bone even for the vertical augmentation when containing a certain amount of plasmid vectors encoding osteogenic proteins. This study supports facilitating the clinical application of GAM for bone engineering.
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http://dx.doi.org/10.1089/biores.2014.0057DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4497668PMC
August 2015

Treatment for salivary gland hypofunction at both initial and advanced stages of Sjögren-like disease: a comparative study of bone marrow therapy versus spleen cell therapy with a 1-year monitoring period.

Cytotherapy 2014 Mar 9;16(3):412-23. Epub 2014 Jan 9.

McGill University, Faculty of Dentistry, Craniofacial Tissue Engineering, and Stem Cells Laboratory, Montreal, Quebec, Canada. Electronic address:

Background Aims: Non-obese diabetic mice (NOD) exhibit autoimmune Sjögren-like disease (SS-like). We reported previously that a combined-therapy consisting of immuno- and cell-based therapy rescued NOD from SS-like. However, therapies tested to date on NOD mice were aimed at the initial phase of SS-like. It is unknown whether therapies are effective in restoring salivary function when given at an advanced phase of SS-like.

Methods: The efficacy of two therapies (bone marrow versus spleen cells) was compared head-to-head for halting/reversing salivary hypofunction at two critical time points of SS-like (7-week-old NOD with normal saliva output and 20-week-old NOD with minimal saliva). NOD mice were divided into four groups: (i) control, (ii) complete Freund's adjuvant (CFA), (iii) bone marrow transplants with CFA or (iv) spleen cell transplants with CFA. Mice were monitored 8-12 months after therapy.

Results: Both cell therapies were effective during the initial phase of SS-like; salivary flow rates were maintained between 80-100% of pre-symptomatic levels. Spleen cell therapy was better than bone marrow when administered in the initial phase of SS-like. When cell therapies were given at an advanced phase of SS-like (20 weeks and older), salivary flow rates improved but were at best 50% of pre-symptomatic levels. Both cell therapies decreased tumor necrosis factor-α, transforming growth factor-β1 levels and T and B cells while increasing epidermal growth factor and regulatory T cells. Elevated serum epidermal growth factor levels were measured in spleen-treated mice.

Conclusions: A therapeutic effect in advanced phase disease, albeit in mice, holds promise for humans in which Sjögren syndrome is generally not diagnosed until a late stage.
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http://dx.doi.org/10.1016/j.jcyt.2013.10.006DOI Listing
March 2014

GDFs promote tenogenic characteristics on human periodontal ligament-derived cells in culture at late passages.

Growth Factors 2013 Oct;31(5):165-73

Salivary Gland Disease Center and Molecular Laboratory for Gene Therapy & Tooth Regeneration, Capital Medical University School of Stomatology , Beijing , China .

Tendon/ligament injures are leading disabilities worldwide. The periodontal ligament (PDL) connects teeth to bone, and is comparable to a tendon/ligament-to-bone insertion. PDL-derived cells (PDLCs) express both osteo/cementogenesis and teno/ligamentogenesis genes. However, an efficient method to induce a tenogenic differentiation of PDLCs has not been thoroughly examined. Therefore, this study tested if growth/differentiation factors (GDFs) enhanced tenogenic characteristics of human PDLCs, as a potential cell source for tendon/ligament engineering. Results demonstrated recombinant GDF-5/GDF-7 inhibited alkaline phosphatase (ALP) activity of PDLCs from passage 3 to 6, while GDF-5 enhanced ALP in dental pulp-derived cells and mesenchymal stem cells. GDF-5 (particularly at 10 ng/ml concentration) induced high expression of both early (scleraxis) and mature (tenomodulin, aggrecan, collagen3) tenogenic genes in P4-6 PDLCs, while inhibiting expression of specific transcription-factors for osteogenic, chondrogenic and adipogenic differentiation. Exogenous GDFs might lead PDLCs being expanded in culture during several passages to highly useful cell source for tendon/ligament engineering.
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http://dx.doi.org/10.3109/08977194.2013.830611DOI Listing
October 2013

Ischemic culture of dental pulp-derived cells is a useful model in which to investigate mechanisms of post-ischemic tissue recovery.

Histol Histopathol 2013 08 30;28(8):985-91. Epub 2013 Apr 30.

Tissue Engineering Research Group, Division of Molecular Therapy, Advanced Clinical Research Center, The Institute of Medical Science, The University of Tokyo, Tokyo, Japan.

Dental pulp is a soft tissue characterized by unique regenerative properties. It is located in the center of each tooth, and is surrounded by hard tissue (dentin). Vascular access is limited to a small foramen at the root apex. Because of this anatomical limitation, dental pulp can easily lose its blood supply, causing the tissue to become ischemic. This occurs, for example, when a tooth is dislocated by traumatic injury or is subjected to inflammation. Since ischemia is caused by a critical shortage of oxygen and nutrients, ischemic damage is usually irreversible, even when the ischemic event is transient. However, unlike ischemia-sensitive organs such as the brain and heart, dental pulp is relatively ischemia-resistant, and recovers from ischemic injury by regenerating damaged tissue. The mechanisms by which this regeneration occurs are poorly understood, but are being investigated in cell culture models that mimic in vivo ischemic conditions using a combination of hypoxia and nutrient deprivation. Here, we review the use of ischemic cell culture to investigate the mechanisms of post-ischemic dental pulp tissue recovery.
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http://dx.doi.org/10.14670/HH-28.985DOI Listing
August 2013

Effect of platelet-rich plasma on bone engineering with an alloplastic substitute containing BMP2.

Biomed Mater Eng 2013 ;23(3):163-72

Department of Oral Surgery, Kamagaya General Hospital, Chiba, Japan.

A robust method for inducing bone-formation without an autograft has not been established. Currently, both platelet-rich plasma (PRP) and bone morphogenetic protein (BMP) have been widely investigated for their clinical use in such cases. However, their synergistic effect is still controversial and previously shown diversity of this effect depends on various factors such as the bone substitutes involved. In this study, we investigated the synergistic effect of PRP and BMP2 on an alloplastic substitute as potentiators to induce in vivo bone-formation. A 10 mm diameter bony defect in rabbit calvarium was reconstructed using biphasic calcium phosphate (BCP) ceramics with or without PRP, recombinant human (rh) BMP2, and their combination. At 6 and 12 weeks after implantation, rabbits were euthanized and the radiographic and histomorphometric features of bone reconstruction were analyzed. The results showed that defects filled by rhBMP2/BCP with or without PRP had high bone density at 6 and 12 weeks in radiological evaluation. However, in histomorphometric analysis, the defects filled by rhBMP2/BCP with PRP showed significant new bone formation compared with that by rhBMP2/BCP without PRP, especially at 6 weeks. We propose that the synergistic effect of PRP and rhBMP2 gives highly osteoinductive properties to alloplastic substitutes in vivo.
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http://dx.doi.org/10.3233/BME-130741DOI Listing
November 2013

Reconstruction of the mandible bone by treatment of resected bone with pasteurization.

J Craniofac Surg 2012 Nov;23(6):1773-5

Department of Oral and Maxillofacial Surgery, Division of Maxillofacial Diagnostic and Surgical Science, Kyushu Dental College, Kitakyushu, Fukuoka, Japan.

The results of long-term follow-up for reimplantation of the mandibular bone treated with pasteurization are reported. Mandibulectomy was performed for mandibular malignancy in 3 cases. The resected bones were subsequently reimplanted after treatment with pasteurization in 3 cases to eradicate tumor cells involved in the resected bone. Although postoperative infection was observed in 2 of 3 cases, reimplantation of the resected mandibular bone treated by pasteurization was finally successful. Ten to 22 years of follow-up was carried out. Pasteurization was able to devitalize tumor cells involved in the resected bone and to preserve bone-inductive activity. Reimplantation of pasteurization could be a useful strategy for reconstruction of the mandible in patients with mandibular malignancy.
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http://dx.doi.org/10.1097/SCS.0b013e318266fd4cDOI Listing
November 2012

In vivo comparison of the bone regeneration capability of human bone marrow concentrates vs. platelet-rich plasma.

PLoS One 2012 12;7(7):e40833. Epub 2012 Jul 12.

Department of Regenerative Oral Surgery, Graduate School of Biomedical Sciences, Nagasaki University, Nagasaki, Japan.

Background: Bone marrow aspirate concentrate (BMAC) including high densities of stem cells and progenitor cells may possess a stronger bone regenerative capability compared with Platelet-rich plasma (PRP), which contains enriched growth factors. The objective of this study was to evaluate the effects of human BMAC and PRP in combination with β-tricalcium phosphate (β-TCP) on promoting initial bone augmentation in an immunodeficient mouse model.

Methodology/principal Findings: BMAC and PRP were concentrated with an automated blood separator from the bone marrow and peripheral blood aspirates. β-TCP particles were employed as a scaffold to carry cells. After cell counting and FACS characterization, three groups of nude mice (BMAC+TCP, PRP+TCP, and a TCP control) were implanted with graft materials for onlay placement on the cranium. Samples were harvested after 4 weeks, and serial sections were prepared. We observed the new bone on light microscopy and performed histomorphometric analysis. After centrifugation, the concentrations of nucleated cells and platelets in BMAC were increased by factors of 2.8 ± 0.8 and 5.3 ± 2.4, respectively, whereas leucocytes and platelets in PRP were increased by factors of 4.1 ± 1.8 and 4.4 ± 1.9, respectively. The concentrations of CD34-, CD271-, CD90-, CD105-, and CD146-positive cells were markedly increased in both BMAC and PRP. The percentage of new bone in the BMAC group (7.6 ± 3.9%) and the PRP group (7.2 ± 3.8%) were significantly higher than that of TCP group (2.7 ± 1.4%). Significantly more bone cells in the new bone occurred in sites transplanted with BMAC (552 ± 257) and PRP (491 ± 211) compared to TCP alone (187 ± 94). But the difference between the treatment groups was not significant.

Conclusions/significance: Both human BMACs and PRP may provide therapeutic benefits in bone tissue engineering applications. These fractions possess a similar ability to enhance early-phase bone regeneration.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0040833PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3395629PMC
January 2013

Characteristic differences among osteogenic cell populations of rat bone marrow stromal cells isolated from untreated, hemolyzed or Ficoll-treated marrow.

Cytotherapy 2012 Aug 12;14(7):791-801. Epub 2012 Apr 12.

Tissue Engineering Research Group, Division of Molecular Therapy, Advanced Clinical Research Center, The Institute of Medical Science, The University of Tokyo, Minato-ku, Tokyo, Japan.

Background Aims: Although bone marrow (BM) stromal cells (SC; BMSC) isolated from adherent cultures of untreated BM are known to contain both committed and uncommitted osteogenic cells, it remains unknown whether BMSC isolated either by hemolysis or Ficoll centrifugation also contain both of these populations.

Methods: Differences in the osteogenic cell populations of rat BMSC isolated from untreated, hemolyzed or Ficoll-treated BM were analyzed by in vivo transplantation, flow cytometry, alkaline phosphatase (ALP) assay, real-time polymerase chain reaction (PCR) and alizarin red staining.

Results: Transplantation of non-cultured samples indicated that the Ficolled BMSC contained the lowest number of committed osteogenic cells. Flow cytometric analysis of cultured, non-induced samples showed that the percentage of ALP-positive cells was significantly lower in Ficolled BMSC. Quantitative ALP assays confirmed that the lowest ALP activity was in the Ficolled BMSC. Hemolyzed BMSC also contained lower numbers of committed osteogenic cells than untreated BMSC, but still more than Ficolled BMSC. Interestingly, the Ficolled BMSC showed the greatest levels of osteogenic ability when cultured in osteogenic induction medium.

Conclusions: These findings suggest that, although Ficolled BMSC rarely contain committed osteogenic cells, they are able to show comparable or even greater levels of osteogenic ability after induction, possibly because they contain a greater proportion of uncommitted stem cells. In contrast, induction is optional but recommended for both untreated and hemolyzed BMSC before use, because both these groups contain both committed and uncommitted osteogenic cells. These findings are of significant importance when isolating BMSC for use in bone tissue engineering.
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http://dx.doi.org/10.3109/14653249.2012.674639DOI Listing
August 2012

Predictive factor for photodynamic therapy effects on oral squamous cell carcinoma and oral epithelial dysplasia.

Arch Oral Biol 2011 Nov 19;56(11):1366-72. Epub 2011 May 19.

Division of Regenerative Oral Surgery, Department of Translational Medicine, Nagasaki University Graduate School of Biomedical Sciences, 1-7-1 Sakamoto, Nagasaki 8528588, Japan.

Objective: The aim of this study was to investigate the correlation between the immunohistochemical expression of proliferating cell nuclear antigen (PCNA), factor VIII, and CD34 (markers of endothelial cells), and vascular endothelial growth factor (VEGF) and the recurrence of oral squamous cell carcinoma (OSCC) and oral epithelial dysplasia (OED) subjected to photodynamic therapy (PDT).

Design: Twenty-one biopsy specimens (14 cases of OSCC and 7 cases of OED) before PDT were immunohistochemically investigated in terms of their expressions of PCNA, factor VIII, CD34 and VEGF. The percentages of the total sample area that were immunopositive for factor VIII (percentage factor VIII immunopositive area: PFIA) CD34 (PCIA) and VEGF (PVIA) were calculated using computer-assisted image analysis for quantitative assessment of endothelial cells or VEGF expression in the lesions. The PCNA labelling index (LI) was evaluated as a proliferation marker.

Results: Five cases of OSCC and one case of OED recurred 4 to 30 months after PDT. We found that the average PVIA was 14.5% in the no-recurrence group and 1.7% in the recurrence group. The difference between these values was statistically significant (P=0.0483). On the other hand, the average PCNA LI was 30.3% in the no-recurrence group and 24.3% in the recurrence group; the average PFIA was 3.7% in the no-recurrence group and 1.6% in the recurrence group; and the average PCIA was 2.0% in the no-recurrence group and 1.4% in the recurrence group. There were no significant differences between the two groups for any of these markers (P=0.3379, P=0.1195, P=0.4835, respectively).

Conclusions: These results provide clinical data indicating that VEGF expression may be a useful predictive marker for the effects of PDT in OSCC and OED.
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http://dx.doi.org/10.1016/j.archoralbio.2011.04.012DOI Listing
November 2011

Bone marrow-derived cells: A potential approach for the treatment of xerostomia.

Int J Biochem Cell Biol 2011 Jan 28;43(1):5-9. Epub 2010 Oct 28.

McGill University, Faculty of Dentistry, Montreal, Canada.

Transplantations of bone marrow-derived cells (BMDCs) are traditionally used for hematologic diseases, but there are increasing numbers of clinical trials using BMDC treatments for non-hematologic disorders, including autoimmune diseases. BMDCs are recently reported to improve organ functions. This paper will review available reports supporting the role of BMDCs in reducing xerostomia (i.e. re-establishing salivary gland functions) due to head and neck irradiation for cancer therapies and in Sjögren's syndrome. There are reports that BMDCs provide a beneficial effect on the saliva production. BMDCs positively affect blood vessels stability and regeneration in irradiated salivary glands. Also, BMDCs provide an immunomodulatory activity in mice with Sjögren's-like disease. While the exact mechanisms by which BMDCs improve organ functions remain controversial, there is preliminary evidence that a combination of them (such as cell transdifferentiation, vasculogenesis, and paracrine effect) occur in salivary glands.
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http://dx.doi.org/10.1016/j.biocel.2010.10.010DOI Listing
January 2011