Plasma Gel Matrix as a Promising Carrier of Epigallocatechin Gallate for Regenerative Medicine.

Plasma gel (PG) is a protein matrix prepared from platelet-poor plasma and can be utilized as a drug carrier for controlled release. We previously demonstrated its applicability as a carrier of polyphosphate. Epigallocatechin-3-gallate (EGCG) is the main flavonoid found in green tea and functions as a strong antioxidant. To explore the applicability of PG as an EGCG carrier, we examined the release of EGCG from the PG matrix using an in vitro system. Pooled platelet-poor plasma (PPP) was prepared from four healthy adult male donors, mixed with EGCG, and heated at 75 °C for 10 or 20 min to prepare the PG matrix. The PG-EGCG matrix was incubated in PBS at 37 °C, and the EGCG released into PBS was determined using spectrophotometry. The antioxidant capacity was determined based on the principle of the iodine decolorization reaction. EGCG precipitated and incorporated into the PG matrix during thermal preparation. Trypsin, used to simulate the in vivo degradation of PG, released EGCG from the PG matrix over time. The released EGCG maintained its antioxidant capacity during incubation. These results indicate that thermally prepared PG matrices can be utilized as a promising EGCG carrier in the fields of tissue engineering and regenerative medicine.

Elevated IL-1ß and Comparable IL-1 Receptor Antagonist Levels Are Characteristic Features of L-PRP in Female College Athletes Compared to Male Professional Soccer Players.

Autologous platelet-rich plasma (PRP) therapy has been becoming popular for the treatment of musculotendinous injuries among athletes. However, for individual and practical variations, clinical success is hardly predictable. To overcome this difficulty, we have been exploring possible criterion candidates for monitoring its clinical effectiveness. In this study, we focused on sex-based differences in young elite athletes and compared the biochemical compositions of their PRP. Leukocyte-rich PRP (L-PRP) was manually prepared from blood samples collected from male professional soccer players (mPSPs) (n = 25) and female college athletes (fCAs) (n = 36). Platelet-derived growth factor-BB (PDGF-BB), transforming-growth factor-ß1 (TGFß1), platelet factor-4 (PF4), interleukin-1ß (IL-1ß), and IL-1 receptor antagonist (IL-1RA) were quantified using an enzyme-linked immunosorbent assay. The levels of PDGF-BB, TGFß1, and PF4 in L-PRP were significantly higher in mPSPs than in fCAs. Conversely, IL-1ß and IL-1RA were detected at significantly and slightly higher levels, respectively, in fCAs than in mPSPs. Our findings suggest that, even though L-PRP from fCAs may have lower potential to induce cell growth and differentiation than that of mPSPs, due to the latter's higher capacity to control inflammation, it does not necessarily imply that PRP treatment in fCAs is less effective. Thus, these cytokine levels should be checked before PRP therapy.

Plasma Gel Made of Platelet-Poor Plasma: In Vitro Verification as a Carrier of Polyphosphate.

Plasma gel (PG) is a blood-derived biomaterial that can be prepared by heating or chemical cross-linking without the aid of intrinsic coagulation activity and has gradually been applied in the field of esthetic surgery. To explore the applicability of PG in regenerative therapy or tissue engineering, in this study, we focused on the advantages of the heating method and verified the retention capacity of the resulting PG for polyphosphate (polyP), a polyanion that contributes to hemostasis and bone regeneration. Pooled platelet-poor plasma (PPP) was prepared from four healthy male adult donors, mixed with synthetic polyP, and heated at 75 °C for 10 or 30 min to prepare PG in microtubes. The PG was incubated in PBS at 37 °C, and polyP levels in the extra-matrix PBS were determined by the fluorometric method every 24 h. The microstructure of PG was examined using scanning electron microscopy. In the small PG matrices, almost all of the added polyP (~100%) was released within the initial 24 h. In contrast, in the large PG matrices, approximately 50% of the polyP was released within the initial 24 h and thereafter gradually released over time. Owing to its simple chemical structure, linear polyP cannot be theoretically retained in the gel matrices used in this study. However, these findings suggest that thermally prepared PG matrices can be applied as carriers of polyP in tissue engineering and regenerative medicine.

Characterization of Leukocyte- and Platelet-Rich Plasma Derived from Female Collage Athletes: A Cross-Sectional Cohort Study Focusing on Growth Factor, Inflammatory Cytokines, and Anti-Inflammatory Cytokine Levels.

Platelet-rich plasma (PRP) has been increasingly used in sports medicine owing to its various advantages. The purpose of our project was to standardize the parameters before performing large-scale clinical trials in the near future to precisely evaluate individual PRP quality. To examine the effects of regular exercise on PRP quality, this study focused on young female athletes, who have been relatively less studied. Blood samples were obtained from female college athletes (n = 35) and ordinary healthy adults (n = 30), which were considered as controls, and leukocyte-rich PRP (L-PRP) was prepared manually. Body composition indices were determined using a bathroom weight scale equipped with an impedance meter. Growth factors and cytokines were quantified using ELISA kits. Platelet-derived growth factor-BB (PDGF-BB) and Transforming-growth factors ß1 (TGFß1) levels (per platelet) in L-PRP were significantly lower in female athletes than in controls. In contrast, Interleukin-1ß and Interleukin 1 receptor antagonist (IL-1RA) levels (per platelet and L-PRP) in L-PRP were significantly higher in athletes, and this difference was more prominent in IL-1RA. These findings suggest that L-PRP from athletes may facilitate the inflammatory phase of the healing process by regulating the pro-inflammatory and anti-inflammatory balance. These chemical compositions can be adopted as "must-check" parameters to characterize individual PRP preparations prior to clinical trials.

Metformin-suppressed platelet's function in vitro: Possible relation to delayed or failure of platelet-rich fibrin preparation.

Platelet-rich fibrin (PRF) is a popular autologous blood-derived biomaterial that is used in regenerative therapy. Owing to its simple preparation without additional factors, the PRF quality directly reflects the characteristics of individual blood samples. Antiplatelet or anticoagulant drugs can hamper the successful preparation of PRF. We recently observed similar phenomena in metformin-taking type-2 diabetics (T2DM). Thus, we hypothesized that metformin interferes with platelet function, thereby suppressing coagulation. For practical reasons, leukocyte- and platelet-rich plasma was prepared from healthy male donors (n = 9-15, age: 26-80 years) and treated with metformin (1-10 mM) for 24-72 h. Intrinsic and extrinsic coagulation activities were evaluated using prothrombin time (PT) and activated partial thromboplastin time (ATPP). Platelet adhesion and aggregation assays were performed using ADP stimulation. Among the parameters tested, APTT was the most sensitive and was significantly prolonged in the concentration range of 1-10 mM in a time- and concentration-dependent manner. Although obtained from healthy platelets and relatively higher concentrations of metformin, these findings suggest that metformin may induce further dysfunction of platelets to suppress intrinsic coagulation activity in T2DM patients, leading to failure of PRF preparation. This phenomenon may not have a severe impact on clinical diabetology or hematology. However, clinicians using PRF are recommended to be more sensitive to such information to avoid unexpected events in clinical settings.

Strategic analysis of body composition indices and resting platelet ATP levels in professional soccer players for better platelet-rich plasma therapy.

Background: Autologous platelet-rich plasma (PRP) therapy is ambiguously thought to be more effective in elite athletes than in sedentary patients, although the possible importance of recipient responsiveness remains poorly understood. To address this issue, along with the well-known PRP quality, in this initial study, we evaluated two candidate biomarkers: body composition indices (BCIs), which reflect systemic physical conditions, and resting platelet ATP levels, which reflect platelet energy expenditure and the mass of energy generation units. Methods: In this cross-sectional cohort study, blood samples were collected from male professional soccer players (PSPs) on a local professional team during the off-season and platelet ATP levels were quantified using an ATP luminescence assay kit. BCIs were measured using the body mass impedance method. Age-matched male sedentary participants were used as the controls. Results: Among the BCIs, the body mass index, basal metabolic rate (BMR), and skeletal muscle weight levels were higher in the PSPs than in the controls. The platelet ATP levels in the PSPs group were significantly lower than those in the control group. The correlation between BMR and platelet ATP levels was moderately negative in the control group, but weakly positive in the PSPs group. Conclusion: Owing to regular physical exercise, PSPs had higher BMR levels and lower platelet ATP levels without a significant mutual correlation compared to sedentary controls. This study did not indicate the influence of these biomarkers on the success of PRP therapy but provided evidence for a better understanding of PRP therapy, particularly for elite athletes.

Optimized Protocol for Preservation of Human Platelet Samples for Fluorometric Polyphosphate Quantification.

Platelet polyphosphate (polyP) can be conveniently quantified by exploiting a recent methodological breakthrough using 4',6-diamidino-2-phenylindole (DAPI). However, the preservation of these biological samples has not yet been standardized. In a preliminary study, potential protocols were screened, while accepted protocols were further tested in this study. Pure-platelet-rich plasma (P-PRP) samples and washed platelet suspensions were prepared using blood obtained from non-smoking healthy male donors and were fixed with ThromboFix for 20-24 h at 4 °C. Mass polyP levels were determined using a fluorometer at wavelengths of 425 and 525 nm. Platelet polyP levels were normalized to platelet counts. Statistical analyses were performed using non-parametric tests. Platelet polyP levels significantly decreased by 20% after 7 days in the platelet suspension maintained under fixed conditions at 4 °C (control). In contrast, the platelet polyP levels in both the P-PRP and washed platelet suspensions were maintained without a significant reduction for up to 6 weeks by removing ThromboFix after fixation and subsequent freezing in pure water at -80 °C. Fluorometric polyP quantification often interferes with the low specificity of DAPI binding and the wavelength used. Our validated protocols will enable long-term preservation and high-throughput polyP quantification and can be applied to relatively large cohort studies.

Standardization of Animal Models and Techniques for Platelet-Rich Fibrin Production: A Narrative Review and Guideline.

Experimental research is critical for advancing medical knowledge and enhancing patient outcomes, including in vitro and in vivo preclinical assessments. Platelet-rich fibrin (PRF) is a blood by-product that has garnered attention in the medical and dental fields due to its potential for tissue regeneration and wound healing. Animal models, such as rabbits and rats, have been used to produce PRF and examine its properties and applications. PRF has demonstrated potential in the dental and medical fields for reducing inflammation, promoting tissue repair, and accelerating wound healing. This narrative review aims to compare existing evidence and provide guidelines for PRF animal research, emphasizing the importance of standardizing animal models, following ethical considerations, and maintaining transparency and accountability. The authors highlight the necessity to use the correct relative centrifugal force (RCF), standardize centrifugal calibration, and report detailed information about blood collection and centrifuge parameters for reproducible results. Standardizing animal models and techniques is crucial for narrowing the gap between laboratory research and clinical applications, ultimately enhancing the translation of findings from bench to bedside.

Modulation of ATP Production Influences Inorganic Polyphosphate Levels in Non-Athletes' Platelets at the Resting State.

Platelets produce inorganic polyphosphate (polyP) upon activation to stimulate blood coagulation. Some researchers have linked polyP metabolism to ATP production, although the metabolic linkage is yet to be elucidated. We found evidence for this possibility in our previous study on professional athletes (versus non-athletes), and proposed that the regulatory mechanism might be different for these two groups. To explore this aspect further, we investigated the effects of modulated ATP production on polyP levels. Blood samples were obtained from Japanese healthy, non-athletes in the presence of acid-citrate-dextrose. The platelets in the plasma were treated with oligomycin, rotenone, and GlutaMAX to modulate ATP production. PolyP level was quantified fluorometrically and visualized using 4',6-diamidino-2-phenylindole. Correlations between polyP and ATP or NADH were then calculated. Contrary to the hypothesis, inhibitors of ATP production increased polyP levels, whereas amino acid supplementation produced the opposite effect. In general, however, polyP levels were positively correlated with ATP levels and negatively correlated with NADH levels. Since platelets are metabolically active, they exhibit high levels of ATP turnover rate. Therefore, these findings suggest that ATP may be involved in polyP production in the resting platelets of non-athletes.

The levels of TGFß1, VEGF, PDGF-BB, and PF4 in platelet-rich plasma of professional soccer players: a cross-sectional pilot study.

BACKGROUND: Regenerative therapy using platelet-rich plasma (PRP), a rich source of growth factors, has become popular in orthopedic sports medicine. Elite athletes prefer PRP therapy for their injured muscles and tendons primarily to avoid the possible risks of surgical treatment. However, the clinical effectiveness of PRP therapy in elite athletes compared to that in non-athletes remains unknown. Therefore, to investigate the effectiveness of PRP therapy in professional athletes (pro-athletes), we focused on the quality of PRP preparations and compared the levels of bioactive molecules between pro-athletes and non-athletes. METHODS: PRP was prepared from healthy, non-smoking male professional soccer players (pro-athletes) (n = 22) and non-athletes (VEGF: n = 34, others: n = 38). The levels of TGFß1, PDGF-BB, VEGF, and PF4 were determined using ELISA kits. Polyphosphate was probed with 4',6-diamidino-2-phenylindole and monitored using a fluorometer. The body composition of the donors was determined using a bathroom weighing scale. RESULTS: The levels of TGFß1 and VEGF were significantly lower in pro-athletes than in non-athletes, whereas PF4 levels were significantly higher in pro-athletes. No significant difference was found in PDGF-BB levels between these groups. Biomolecule levels were not correlated with polyphosphate levels. CONCLUSION: TGFß1, VEGF, and PDGF-BB levels in pro-athletes were not higher than those in non-athletes. These findings suggest that growth factor levels in PRP may not be a predominant determinant of the clinical effectiveness of PRP therapy in pro-athletes. Increased PF4 levels in pro-athletes suggest an immunological function of PRP that may positively influence tissue regeneration.

Platelet polyphosphate and energy metabolism in professional male athletes (soccer players): A cross-sectional pilot study.

Human platelet polyphosphate (polyP) is a multifunctional molecule; however, its functions are not yet fully understood. A recent study demonstrated that similar to skeletal muscle, polyP is involved in energy metabolism in platelets, which suggests that well-trained athletes may exhibit elevated platelet polyP levels for energy storage. To test this hypothesis, we quantified platelet polyP along with NADH, a component involved in ATP production in non-trained and well-trained male Japanese participants of the same generation. Washed platelets were prepared from the venous blood of young, healthy, non-athletes, and professional soccer players (pro-athletes). NADH and polyP levels were spectrophotometrically determined using tetrazolium reduction and fluorometrically determined using 4',6-diamidino-2-phenylindole at the excitation/emission wavelengths of 425/525 nm. Body weight and impedances were measured simultaneously. Statistical analyses were performed using the Mann-Whitney U test and Spearman correlation coefficient. Although basal metabolic rate levels were significantly higher, platelet polyP levels were significantly lower in pro-athletes than in that in non-athletes. No significant differences were detected in other body compositions or platelet indices between the two groups. The pro-athlete group showed a moderate, nearly significant correlation (R = 0.439; p = 0.0512) between platelet polyP and NADH levels. Taken together with the weak correlation data between polyP and body mass index, it is suggested that platelet polyP levels may be influenced by platelet and body energy metabolic activity. Further biochemical studies are needed to elucidate this mechanism.

Responses of promyelocytic leukemia HL60 cells as an inflammatory cell lineage model to silica microparticles used to coat blood collection tubes.

BACKGROUND: The preparation of platelet-rich fibrin (PRF) requires glass blood collection tubes, and thus, the shortage or unavailability of such tubes has driven clinicians to search for suitable substitutes, such as silica-coated plastic tubes. However, we have previously demonstrated the cytotoxicity of silica microparticles (MPs) used in plastic tubes to cultured human periosteal cells. To further establish the effects of silica MPs on inflammation, we examined silica MP-induced changes in a human promyelocytic cell model in vitro. METHODS: Human promyelocytic HL60 cells were used either without chemical induction or after differentiation induced using phorbol myristate acetate (PMA) or dimethyl sulfoxide. HL60 cells, osteoblastic MG63, and Balb/c mouse cells were treated with silica MPs, and their surface ultrastructure and numbers were examined using a scanning electron microscope and an automated cell counter, respectively. Differentiation markers, such as acid phosphatase, non-specific esterase, and CD11b, were visualized by cytochemical and immunofluorescent staining, and superoxide dismutase (SOD) activity was quantified. RESULTS: Regardless of SOD activity, silica cytotoxicity was observed in MG63 and Balb/c cells. At sub-toxic doses, silica MPs slightly or moderately upregulated the differentiation markers of the control, PMA-induced monocytic, and dimethyl sulfoxide-induced granulocytic HL60 cells. Although SOD activity was the highest (P < 0.05) in PMA-induced cells, a silica-induced reduction in cell adhesion was observed only in those cells (P < 0.05). CONCLUSIONS: Silica MP contamination of PRF preparations can potentially exacerbate inflammation at implantation sites. Consequently, unless biomedical advantages can be identified, silica-coated plastic blood collection tubes should not be routinely used for PRF preparations.

Non-destructive, spectrophotometric analysis of the thickness of the cell-multilayered periosteal sheet.

BACKGROUND: Autologous tissue-engineered periosteal sheets, which have been clinically applied for periodontal regeneration, sinus lift, and alveolar ridge augmentation, are enriched with osteoblast precursor cells and the abundant deposition of collagen type I in the extracellular spaces. Their quality is inspected prior to clinical use; however, most criteria cannot be evaluated without sacrificing samples. To reduce such losses, we developed a non-destructive optical method that can quantitatively evaluate the thickness of the periosteal sheet. METHODS: Dispersed periosteal cells were inoculated into small pieces of collagen sponge (Terudermis®) and plated into 60-mm dishes for further explant culture using a conventional medium and a stem-cell culture medium. The thickness of periosteal sheets was evaluated using inverted microscopic, histological, labeling (CellVue®)-based imaging and spectrophotometric (Spectro-1®) methods. RESULTS: The three-dimensional growth of periosteal sheets did not necessarily correlate with two-dimensional growth. The periosteal sheet prepared with the stem-cell medium formed cell multilayers, a phenomenon that could be observed qualitatively by inverted microscopy. The spectrophotometric analysis enabled the quantitative evaluation of the thickness of the cell multilayer without sacrificing the samples processed for scheduled cell therapy. CONCLUSIONS: The growth of periosteal sheets is influenced by several major factors, including the basic quality of the individual original periosteal tissue segments, the technical expertise of doctors and operators involved in tissue harvesting and processing, and culture conditions. This newly developed spectrophotometric analysis can quantify the thickness of cell-multilayered periosteal sheets for quality assurance in a non-destructive manner, thereby contributing to better bone augmentation prior to implant therapy.

A Strategic and Worldwide Cooperative Challenge Required for the Next Generation of Platelet Concentrates.

Blood-derived biomaterials, which are represented by platelet-rich plasma (PRP) or more recently by platelet-rich fibrin (PRF), have been used in regenerative therapy for almost 30 years [...].

Effects of SARS-CoV-2 mRNA vaccines on platelet polyphosphate levels and inflammation: A pilot study.

Platelets function as immune cells in conjunction with white blood cells, targeting invading pathogens and inducing immune reactions. Intercellular communications among these immune cells are partly mediated by platelet polyphosphate (polyP), which was originally recognized as a thrombotic and hemostatic biomolecule. To determine the involvement of polyP in SARS-CoV-2-mRNA vaccine-induced immune responses, specifically in inflammatory responses, the effects of mRNA vaccines on platelet polyP levels were examined. Before and after vaccination with the COVID-19 vaccine (BNT162b2), blood samples were obtained from healthy, non-smoking individuals who did not have any systemic diseases. Test group demographics skewed somewhat towards either older males (first vaccination, n=6; second vaccination, n=8) or younger females (first vaccination, n=14; second vaccination, n=23). polyP levels in washed platelets from the blood samples were determined using the fluorometric method with DAPI. The side-effects of vaccination were recorded as scores. In the female group, platelet polyP levels decreased after the first vaccination, and the side-effect score increased after the second vaccination. Moderate correlation coefficients were observed between the reduction in polyP levels and the side-effect scores and pre-vaccination polyP levels. Despite the small sample size, this pilot study suggests that platelet polyP may suppress the side effects induced by the mRNA vaccines after the first vaccination, but not the second vaccination in younger female subjects, who generally have higher immune responsiveness than their male counterparts.

Distribution and quantification of activated platelets in platelet-rich fibrin matrices.

Platelet-rich fibrin (PRF) has been widely applied in regenerative therapy owing to its simple preparation protocol. To date, the original protocol for preparing leukocyte-rich (L)-PRF has been modified to produce derivatives such as advanced (A)-PRF, concentrated growth factors (CGF), and horizontal (H)-PRF. However, these derivatives have not been rigorously compared to explore possible differences. We previously developed and validated a nondestructive near-infrared (NIR) imaging method to quantitatively examine the platelet distribution in PRF matrices. To further evaluate the characteristics of platelets in PRF, we herein examined the distribution of activated platelets. Four types of PRF matrices were prepared under different centrifugal conditions from blood samples obtained from the same healthy donors. After fixation and compression, the matrices were stained immunohistochemically without sectioning and visualized using an NIR imager. Qualitative morphological analysis revealed that whole platelets were distributed widely and homogeneously in H-PRF and A-PRF, but localized along the distal tube surface in L-PRF and CGF. Activated platelets were distributed as were whole platelets in A-PRF, L-PRF, and CGF, but localized mainly in the "buffy coat" region in H-PRF. Quantitative analysis revealed that there was no significant difference in the ratio of activated to whole platelets between PRF derivatives. These findings suggest that platelet activation is similarly induced in fibrin matrices regardless of centrifugal speed or rotor angulation. However, only the H-PRF group was distinguishable from the other PRF derivatives in terms of activated platelet distribution.

Histological Evaluation of Gingival and Intrabony Periodontal Defects Treated with Platelet-rich Fibrin Using Different Protocols: A Canine Study.

PURPOSE: To histologically compare the effects of platelet-rich fibrin (PRF) produced using different protocols on periodontal wound healing/regeneration in periodontal defects in dogs. MATERIALS AND METHODS: Dehiscence-type gingival recession and two-wall intrabony defects were created bilaterally in the maxillary canines and mandibular premolars, respectively, in four beagle dogs. The recession defects were randomly treated with coronally advanced flap (CAF) alone, CAF and PRF produced via fixed-angle centrifugation (F-PRF; Leukocyte and PRF (L-PRF) protocol) or CAF and PRF produced via horizontal centrifugation (H-PRF). After 2 weeks, the two-wall intrabony defects were randomly treated as follows: open flap debridement (OFD), OFD + F-PRF, OFD + H-PRF and OFD + heated albumin with PRF using bio-heat technologies (Alb-PRF). Eight weeks after the 2nd reconstructive surgery, the animals were euthanised for histological evaluation. RESULTS: In the PRF-applied defects, new bone and new cementum formation occurred to varying degrees regardless of the protocols used to produce PRF. Particularly in the two-wall intrabony defects, new bone formation extended from the host bone toward the coronal region of the defects in the H-PRF applied sites compared with those in the OFD, F-PRF and Alb-PRF groups, and the H-PRF group showed the greatest amount of newly formed cementum. CONCLUSION: PRF induced periodontal regeneration in gingival recession and two-wall intrabony defects in dogs. Further studies are needed to determine the optimal protocol for obtaining predictable periodontal regeneration in periodontal defects in humans.

Fluorometric Quantification of Human Platelet Polyphosphate Using 4',6-Diamidine-2-phenylindole Dihydrochloride: Applications in the Japanese Population.

Polyphosphate (polyP), a biopolymer of inorganic phosphate, is widely distributed in living organisms. In platelets, polyP is released upon activation and plays important roles in coagulation and tissue regeneration. However, the lack of a specific quantification method has delayed the in-depth study of polyP. The fluorescent dye 4',6-diamidine-2-phenylindole dihydrochloride (DAPI) has recently received attention as a promising probe for the visualization and quantification of cellular polyP levels. In this study, we further optimized quantification conditions and applied this protocol in quantification of platelet polyP levels in a Japanese population. Blood samples were collected from non-smoking, healthy Japanese subjects (23 males, 23 females). Washed platelets were fixed and probed with DAPI for fluorometric determination. PolyP levels per platelet count were significantly higher in women than that in men. A moderate negative correlation between age and polyP levels was found in women. Responsiveness to CaCl2 stimulation was also significantly higher in women than that in men. Overall, our optimized protocol requires neither purification nor degradation steps, reducing both the time and bias for reproducible quantification. Thus, we suggest that despite its low specificity, this DAPI-based protocol would be useful in routine laboratory testing to quantify platelet polyP levels efficiently and economically.

Osteoclastogenic Potential of Tissue-Engineered Periosteal Sheet: Effects of Culture Media on the Ability to Recruit Osteoclast Precursors.

Cell culture media influence the characteristics of human osteogenic periosteal sheets. We have previously found that a stem cell medium facilitates growth and collagen matrix formation in vitro and osteogenesis in vivo. However, it has not yet been demonstrated which culture medium is superior for osteoclastogenesis, a prerequisite for reconstruction of normal bone metabolic basis. To address this question, we compared chemotaxis and osteoclastogenesis in tissue-engineered periosteal sheets (TPSs) prepared with two types of culture media. Periosteal tissues obtained from adult volunteers were expanded with the conventional Medium 199 or with the stem cell medium, MesenPRO. Hematopoietic enhanced-green-fluorescent-protein (EGFP)-nude mice were prepared by γ-irradiation of Balb/c nu/nu mice and subsequent transplantation of bone marrow cells from CAG-EGFP C57BL/6 mice. TPSs were implanted subcutaneously into the chimeric mice and retrieved after intervals for immunohistopathological examination. EGFP+ cells were similarly recruited to the implantation site in both the TPSs prepared, whereas the distribution of CD11b+ cells was significantly lower in the TPS prepared with the stem cell medium. Instead, osteoclastogenesis was higher in the TPS prepared with the stem cell medium than in the one prepared with the conventional medium. These findings suggest that the stem cell medium is preferable for the preparation of more functional TPSs.