Kohya Uematsu

A clinical study of alveolar bone tissue engineering with cultured autogenous periosteal cells: Coordinated activation of bone formation and resorption

In ongoing clinical research into the use of cultured autogenous periosteal cells (CAPCs) in alveolar bone regeneration, CAPCs were grafted into 33 sites (15 for alveolar ridge augmentation and 18 for maxillary sinus lift) in 25 cases. CAPCs were cultured for 6weeks, mixed with particulate autogenous bone and platelet-rich plasma, and then grafted into the sites. Clinical outcomes were determined from high-resolution three-dimensional computed tomography (3D-CT) images and histological findings. No serious adverse events were attributable to the use of grafted CAPCs. Bone regeneration was satisfactory even in cases of advanced atrophy of the alveolar process. Bone biopsy after bone grafting with CAPCs revealed prominent recruitment of osteoblasts and osteoclasts accompanied by angiogenesis around the regenerated bone. 3D-CT imaging suggested that remodeling of the grafted autogenous cortical bone particles was faster in bone grafting with CAPCs than in conventional bone grafting. The use of CAPCs offers cell-based bone regeneration therapy, affording complex bone regeneration across a wide area, and thus expanding the indications for dental implants. Also, it enables the content of particulate autogenous bone in the graft material to be reduced to as low as 40%, making the procedure less invasive, or enabling larger amounts of graft materials to be prepared. It may also be possible to dispense with the use of autogenous bone altogether in the future. The results suggest that CAPC grafting induces bone remodeling, thereby enhancing osseointegration and consequently reducing postoperative waiting time after dental implant placement.

Growth factor and pro-inflammatory cytokine contents in platelet-rich plasma (PRP), plasma rich in growth factors (PRGF), advanced platelet-rich fibrin (A-PRF), and concentrated growth factors (CGF)

BackgroundThe development of platelet-rich fibrin (PRF) drastically simplified the preparation procedure of platelet-concentrated biomaterials, such as platelet-rich plasma (PRP), and facilitated their clinical application. PRF’s clinical effectiveness has often been demonstrated in pre-clinical and clinical studies; however, it is still controversial whether growth factors are significantly concentrated in PRF preparations to facilitate wound healing and tissue regeneration. To address this matter, we performed a comparative study of growth factor contents in PRP and its derivatives, such as advanced PRF (A-PRF) and concentrated growth factors (CGF).MethodsPRP and its derivatives were prepared from the same peripheral blood samples collected from healthy donors. A-PRF and CGF preparations were homogenized and centrifuged to produce extracts. Platelet and white blood cell counts in A-PRF and CGF preparations were determined by subtracting those counts in red blood cell fractions, supernatant acellular serum fractions, and A-PRF/CGF exudate fractions from those counts of whole blood samples. Concentrations of growth factors (TGF-β1, PDGF-BB, VEGF) and pro-inflammatory cytokines (IL-1β, IL-6) were determined using ELISA kits.ResultsCompared to PRP preparations, both A-PRF and CGF extracts contained compatible or higher levels of platelets and platelet-derived growth factors. In a cell proliferation assay, both A-PRF and CGF extracts significantly stimulated the proliferation of human periosteal cells without significant reduction at higher doses.ConclusionsThese data clearly demonstrate that both A-PRF and CGF preparations contain significant amounts of growth factors capable of stimulating periosteal cell proliferation, suggesting that A-PRF and CGF preparations function not only as a scaffolding material but also as a reservoir to deliver certain growth factors at the site of application.

Tissue culture of human alveolar periosteal sheets using a stem-cell culture medium (MesenPRO-RS™): In vitro expansion of CD146-positive cells and concomitant upregulation of osteogenic potential in vivo

We have previously demonstrated that multilayered periosteal sheets prepared from the explant culture of alveolar periosteum serve as a promising osteogenic grafting material in periodontal tissue regeneration. For the preparation of more potent periosteal sheets, we examined the applicability of stem-cell culture media. Compared to the control medium (Medium 199+10% FBS), periosteal sheets expanded with MesenPRO-RS™ medium exhibited these features: Cells grew three-dimensionally and deposited collagen in the extracellular spaces to form thicker multilayers of cells. Chondrocytic markers were not significantly upregulated. Contractile force was generated in proportion with the increased thickness of the periosteal sheets and the formation of cytoplasmic α-smooth muscle actin fibers. However, myofibroblastic markers were not significantly upregulated. The surface marker CD146 was substantially upregulated, while both CD73 and CD105 were downregulated. Alkaline phosphatase, a representative osteoblastic marker, was not upregulated by osteogenic induction. However, these expanded periosteal sheets exhibited substantially stronger osteogenic differentiation when implanted in nude mice. Therefore, despite our reservations, MesenPRO medium effectively expanded the cells contained in periosteal sheets to promote the formation of thicker multilayers of cells in vitro, and these enhanced periosteal sheets expressed increased osteogenic potential at implantation sites in vivo. In conjunction with data indicating that CD146-positive cells were notably expanded and the recently proposed concept that CD146 is a marker for osteogenic progenitor cells found in the bone marrow stroma, our findings suggest that MesenPRO medium improves the preparation of highly osteogenic periosteal sheets suitable for clinical application largely through the induction of CD146-positive cells.

ITGA3 and ITGB4 expression biomarkers estimate the risks of locoregional and hematogenous dissemination of oral squamous cell carcinoma

BackgroundMolecular biomarkers are essential for monitoring treatment effects, predicting prognosis, and improving survival rate in oral squamous cell carcinoma. This study sought to verify the effectiveness of two integrin gene expression ratios as biomarkers.MethodsGene expression analyses of integrin α3 (ITGA3), integrin β4 (ITGB4), CD9 antigen (CD9), and plakoglobin (JUP) by quantitative real-time PCR were conducted on total RNA from 270 OSCC cases. The logrank test, Cox proportional hazards model, and Kaplan-Meier estimates were performed on the gene expression ratios of ITGA3/CD9 and ITGB4/JUP and on the clinicopathological parameters for major clinical events.ResultsA high rate (around 80%) of lymph node metastasis was found in cases with a high ITGA3/CD9 ratio (high-ITGA3/CD9) and invasive histopathology (YK4). Primary site recurrence (PSR) was associated with high-ITGA3/CD9, T3-4 (TNM class), and positive margin, indicating that PSR is synergistically influenced by treatment failure and biological malignancy. A high ITGB4/JUP ratio (high-ITGB4/JUP) was revealed to be a primary contributor to distant metastasis without the involvement of clinicopathological factors, suggesting intervention of a critical step dependent on the function of the integrin β4 subunit. Kaplan-Meier curves revealed positive margin as a lethal treatment consequence in high-ITGA3/CD9 and YK4 double-positive cases.ConclusionTwo types of metastatic trait were found in OSCC: locoregional dissemination, which was reflected by high-ITGA3/CD9, and distant metastasis through hematogenous dissemination, uniquely distinguished by high-ITGB4/JUP. The clinical significance of the integrin biomarkers implies that biological mechanisms such as cancer cell motility and anchorage-independent survival are vital for OSCC recurrence and metastasis.

An Evaluation of the Accuracy of the Subtraction Method Used for Determining Platelet Counts in Advanced Platelet-Rich Fibrin and Concentrated Growth Factor Preparations

Platelet concentrates should be quality-assured of purity and identity prior to clinical use. Unlike for the liquid form of platelet-rich plasma, platelet counts cannot be directly determined in solid fibrin clots and are instead calculated by subtracting the counts in other liquid or semi-clotted fractions from those in whole blood samples. Having long suspected the validity of this method, we herein examined the possible loss of platelets in the preparation process. Blood samples collected from healthy male donors were immediately centrifuged for advanced platelet-rich fibrin (A-PRF) and concentrated growth factors (CGF) according to recommended centrifugal protocols. Blood cells in liquid and semi-clotted fractions were directly counted. Platelets aggregated on clot surfaces were observed by scanning electron microscopy. A higher centrifugal force increased the numbers of platelets and platelet aggregates in the liquid red blood cell fraction and the semi-clotted red thrombus in the presence and absence of the anticoagulant, respectively. Nevertheless, the calculated platelet counts in A-PRF/CGF preparations were much higher than expected, rendering the currently accepted subtraction method inaccurate for determining platelet counts in fibrin clots. To ensure the quality of solid types of platelet concentrates chairside in a timely manner, a simple and accurate platelet-counting method should be developed immediately.

Manual cryopreservation of human alveolar periosteal tissue segments: Effects of pre-culture on recovery rate

Cultured human periosteal sheets constitute a promising grafting material for periodontal tissue regenerative therapy. However, preparation of these sheets usually requires six weeks or longer, and this lengthy commitment and delay limits both clinical applicability and availability. The aim of this study is to develop an efficient, practical, cost-effective cryopreservation method for periosteal tissue segments (PTSs). Human PTSs were aseptically excised from alveolar bone and pre-cultured in Medium 199+10% fetal bovine serum (FBS) for the indicated number of days before they were slowly frozen down to -75°C in a commercial freezing vessel using medium containing 10% dimethyl sulfoxide (Me(2)SO) and various concentrations of FBS. After fast-thawing at 37°C, PTSs were again cultured, and their growth and responses to standard osteogenic induction were evaluated (vs. freshly excised PTSs). Proliferating cells were obtained at the highest levels from cryopreserved PTSs that were pre-cultured for 14 days before freezing. When a concentration of 50% or more FBS was included in the cryopreservation solution, cells migrated out more actively and grew faster. Importantly, osteoinduction up-regulated alkaline phosphatase (ALP) activity and osteoblastic marker mRNAs in cryopreserved PTS-derived sheets just as effectively as it did in native PTS-derived ones. These data suggest that pre-conditioned PTSs can be efficiently cryopreserved in a freezing solution containing high FBS by traditional manual cryopreservation methods without aid of a program freezer or more elaborate equipment.

Platelet Counts in Insoluble Platelet-Rich Fibrin Clots: A Direct Method for Accurate Determination

Platelet-rich fibrin (PRF) clots have been used in regenerative dentistry most often, with the assumption that growth factor levels are concentrated in proportion to the platelet concentration. Platelet counts in PRF are generally determined indirectly by platelet counting in other liquid fractions. This study shows a method for direct estimation of platelet counts in PRF. To validate this method by determination of the recovery rate, whole-blood samples were obtained with an anticoagulant from healthy donors, and platelet-rich plasma (PRP) fractions were clotted with CaCl2 by centrifugation and digested with tissue-plasminogen activator. Platelet counts were estimated before clotting and after digestion using an automatic hemocytometer. The method was then tested on PRF clots. The quality of platelets was examined by scanning electron microscopy and flow cytometry. In PRP-derived fibrin matrices, the recovery rate of platelets and white blood cells was 91.6 and 74.6%, respectively, after 24 h of digestion. In PRF clots associated with small and large red thrombi, platelet counts were 92.6 and 67.2% of the respective total platelet counts. These findings suggest that our direct method is sufficient for estimating the number of platelets trapped in an insoluble fibrin matrix and for determining that platelets are distributed in PRF clots and red thrombi roughly in proportion to their individual volumes. Therefore, we propose this direct digestion method for more accurate estimation of platelet counts in most types of platelet-enriched fibrin matrix.

Basic characteristics of plasma rich in growth factors (PRGF): blood cell components and biological effects

Platelet‐rich plasma (PRP) is widely used in regenerative medicine because of its high concentrations of various growth factors and platelets. However, the distribution of blood cell components has not been investigated in either PRP or other PRP derivatives. In this study, we focused on plasma rich in growth factors (PRGF), a PRP derivative, and analyzed the distributions of platelets and white blood cells (WBCs). Peripheral blood samples were collected from healthy volunteers (N = 14) and centrifuged to prepare PRGF and PRP. Blood cells were counted using an automated hematology analyzer. The effects of PRP and PRGF preparations on cell proliferation were determined using human periosteal cells. In the PRGF preparations, both red blood cells and WBCs were almost completely eliminated, and platelets were concentrated by 2.84‐fold, whereas in the PRP preparations, both platelets and WBCs were similarly concentrated by 8.79‐ and 5.51‐fold, respectively. Platelet counts in the PRGF preparations were positively correlated with platelet counts in the whole blood samples, while the platelet concentration rate was negatively correlated with red blood cell counts in the whole blood samples. In contrast, platelet counts and concentration rates in the PRP preparations were significantly influenced by WBC counts in whole blood samples. The PRP preparations, but not the PRGF preparations, significantly suppressed cell growth at higher doses in vitro. Therefore, these results suggest that PRGF preparations can clearly be distinguished from PRP preparations by both inclusion of WBCs and dose‐dependent stimulation of periosteal cell proliferation in vitro.

Application of stem-cell media to explant culture of human periosteum: An optimal approach for preparing osteogenic cell material:

As part of our clinical tests on bone regeneration using cultured periosteal sheets, here, we prepared cultured periosteal sheets in two types of stem-cell culture media, STK1 and STK3. Human periosteum was expanded either in 1% human serum–supplemented STK1 for 28 days, in 1% human serum–supplemented STK1 for 14 days followed by 1% human serum–supplemented STK3 for 14 days (1% human serum–supplemented STK1+3), or in 10% fetal bovine serum–supplemented Medium 199 for 28 days (control). Cultured periosteal sheet diameter and DNA content were significantly higher, and the multilayer structure was prominent in 1% human serum–supplemented STK1 and 1% human serum–supplemented STK1+3. The messenger RNA of osteoblastic markers was significantly upregulated in 1% human serum–supplemented STK1+3. Osteopontin-immunopositive staining and mineralization were evident across a wide area of the cultured periosteal sheet in 1% human serum–supplemented STK1+3. Subcutaneous implantation in nude mice following expansion in 1% human serum–supplemented STK1+3 produced the highest cultured periosteal sheet osteogenic activity. Expansion in 1% human serum–supplemented STK1+3 successfully induced cultured periosteal sheet growth while retaining osteogenic potential, and subsequent osteoblastic induction promoted the production of homogeneous cell material.

High‐Resolution Three‐Dimensional Computed Tomography Analysis of the Clinical Efficacy of Cultured Autogenous Periosteal Cells in Sinus Lift Bone Grafting

Abstract Background and Purpose Sinus lift (SL) using cultured autogenous periosteal cells (CAPCs) combined with autogenous bone and platelet‐rich plasma (PRP) was performed to evaluate the effect of cell administration on bone regeneration, by using high‐resolution three‐dimensional computed tomography (CT). Materials and Methods SL with autogenous bone and PRP plus CAPC [CAPC(+)SL] was performed in 23 patients. A piece of periosteum taken from the mandible was cultured in M199 medium with 10% fetal bovine serum (FBS) for 6 weeks. As control, 16 patients received SL with autogenous bone and PRP [CAPC(−)SL]. Three‐dimensional CT imaging was performed before and 4 months and 1 year after SL, and stratification was performed based on CT numbers (HUs) corresponding to soft tissue and cancellous or cortical bone. Results The augmented bone in CAPC(+)SL revealed an increase in HUs corresponding to cancellous bone as well as a decrease in HUs corresponding to grafted cortical bone. In addition, HUs corresponding to cancellous bone in the graft bed were increased in CAPC(+)SL but were decreased in CAPC(−)SL. Insertion torque during implant placement was significantly higher in CAPC(+)SL. Conclusion By promoting bone anabolic activity both in augmented bone and graft bed, CAPCs are expected to aid primary fixation and osseointegration of implants in clinical applications.