Three Milliliters of Peripheral Blood Is Sufficient for Preparing Liquid Platelet-Rich Fibrin (PRF): An In Vitro Study.

Platelet-rich fibrin (PRF) has assumed an important role in supporting tissue regeneration in different fields. To date, the standard protocol for liquid PRF requires at least 10 mL of peripheral blood. The present study aimed to analyze the composition, growth factor release, and effects on the cell proliferation of PRF samples produced using 3 mL vs. 10 mL of peripheral blood in vitro. Peripheral venous blood from six healthy donors was used to prepare liquid PRF using either 3 mL or 10 mL tubes. Three different centrifugation protocols were used according to the low-speed centrifugation concept. The cellular distribution was evaluated using immunohistology and automated cell count. ELISA was used to determine the release of different growth factors (EGF, TGF-ß1, and PDGF) and interleukin 8 at different time points. Primary human osteoblasts (pOBs) were cultivated for 7 days using PRF-conditioned media acquired from either 3 mL or 10 mL of peripheral blood. The results showed that 3 mL of peripheral blood is sufficient to produce a liquid PRF concentrate similar to that acquired when using 10 mL blood. The concentrations of platelets and leukocytes were comparable regardless of the initial blood volume (3 mL vs. 10 mL). Similarly, the release of growth factors (EGF, TGF-ß1, and PDGF) and interleukin 8 was often comparable in both groups over 7 days. The cultivation of pOBs using PRF-conditioned media showed a similar proliferation rate regardless of the initial blood volume. This proliferation rate was also similar to that of pOBs treated with 20% FBS-conditioned media. These findings validated the use of 3 mL of peripheral blood to generate liquid PRF matrices according to the low-speed centrifugation concept, which may open new application fields for research purposes such as in vivo experiments and clinical applications such as pediatric surgery.

Effectiveness of Oroantral Communication Closure Using Solid Platelet-Rich Fibrin Compared to a Conventional Treatment Approach: A Randomized Clinical Trial.

This study aimed to compare the effectiveness of platelet-rich fibrin clot (PRF) and conventional oroantral communication (OAC) repair techniques following tooth extractions from the maxilla. The study involved 22 patients, divided into 2 groups: a study group treated with PRF and a control group undergoing conventional OAC repair. The primary outcome measured was the effectiveness of OAC closure, with pain intensity and postoperative complications as secondary outcomes. Cone-beam computed tomography (CBCT) was used to evaluate post-extraction bone regeneration. Pain was assessed using the Visual Analogue Scale (VAS) on days 0, 1, 7, and 14 postoperatively. The results showed that the PRF group experienced a significant decrease in pain within the first 24 hours and after 7 days (P < .0001; P < .05). In contrast, complications were reported in 45.45% of patients in the conventional repair group and 18.18% in the A-PRF group. Three months postsurgery, CBCT revealed appreciable bone healing in both groups, with no significant difference (P > .05). In conclusion, the study suggests that A-PRF treatment for OACs resulted in fewer complications and quicker pain reduction than traditional repair methods, making it a promising alternative for managing OACs. However, future studies are needed to confirm these findings and establish the full therapeutic potential of PRF.

Fucoidan/chitosan hydrogels as carrier for sustained delivery of platelet-rich fibrin containing bioactive molecules.

Platelet-rich fibrin (PRF), derived from human blood, rich in wound healing components, has drawbacks in direct injections, such as rapid matrix degradation and growth factor release. Marine polysaccharides, mimicking the human extracellular matrix, show promising potential in tissue engineering. In this study, we impregnated the self-assembled fucoidan/chitosan (FU_CS) hydrogels with PRF obtaining PRF/FU_CS hydrogels. Our objective was to analyze the properties of a hydrogel and the sustained release of growth factors from the hydrogel that incorporates PRF. The results of SEM and BET-BJH demonstrated the relatively porous nature of the FU_CS hydrogels. ELISA data showed that combining FU_CS hydrogel with PRF led to a gradual 7-day sustained release of growth factors (VEGF, EGF, IL-8, PDGF-BB, TGF-ß1), compared to pure PRF. Histology confirmed ELISA data, demonstrating uniform PRF fibrin network distribution within the FU_CS hydrogel matrix. Furthermore, the FU_CS hydrogels revealed excellent cell viability. The results revealed that the PRF/FU_CS hydrogel has the potential to promote wound healing and tissue regeneration. This would be the first step in the search for improved growth factor release.

Current Knowledge on the Healing of the Extraction Socket: A Narrative Review.

The concept of extraction socket healing has been severally researched and reported over the years, since tooth extraction remains one of the most common procedures performed in the dental clinic. Understanding this healing process is of utmost importance because the outcome has a direct bearing on future prosthetic rehabilitation and, by extension, on patients' esthetics and masticatory function, among others. This mini review, therefore, summarized the current knowledge on the different stages of socket healing, including the biologic and clinical events that occur following tooth extraction up until the complete closure of the socket. Additionally, the modeling of the alveolar bone/process post extraction, and the resultant dimensional changes that, altogether, shape the bone, were reviewed and documented. The effects of various socket preservation interventions to mitigate these dimensional changes, and therefore preserve the alveolar process in a condition suitable for future prosthetic rehabilitation, were highlighted. Finally, a review of some of the factors that influence the entire process was also carried out.

Platelet-Rich Fibrin-Conditioned Medium as an Alternative to Fetal Bovine Serum Promotes Osteogenesis of Human Dental Pulp Stem Cells.

Human dental pulp stem cells (DPSCs) exhibit multilineage differentiation capabilities and superior clonogenic and proliferative properties. However, the use of animal-derived components such as FBS raises concerns regarding the clinical application of stem-cell-based therapies. Platelet-rich fibrin (PRF) derived from human blood is rich in fibrin, platelets, and growth factors and acts as a bioactive scaffold for grafting with biomaterials. In this study, we assessed the efficacy of PRF-conditioned medium (CM) in promoting DPSCs proliferation and osteogenic differentiation compared with the standard culture medium supplemented with FBS. A comparison of DPSCs cultured in FBS and PRF-CM revealed no differences in characteristics or morphology. However, cells cultured with PRF-CM exhibited inferior proliferation rates and cell numbers during passage in comparison with those cultured with FBS. In contrast, DPSCs cultured in PRF-CM showed significantly higher levels of calcification, and RT-PCR confirmed that the gene expression levels of markers associated with osteoblast differentiation were significantly increased. The PRF-CM approach offers a convenient, straightforward, and advantageous method for culturing DPSCs, without relying on animal-derived components. In summary, this study introduces a novel application of PRF-CM for enhancing the osteogenesis of DPSCs, which provides an alternative to FBS culture medium and addresses concerns associated with the use of animal-derived components in clinical settings.

Hyperspectral Imaging Allows Evaluation of Free Flaps in Craniomaxillofacial Reconstruction.

INTRODUCTION: Treatment of extended defects after tumor resection in oral, maxillofacial, and facial surgery (craniomaxillofacial) is usually performed by free microvascular flaps. Evaluation of flap survival is crucial, especially in the first hours after insertion and connection. For flap evaluation various invasive and noninvasive methods have been developed. This retrospective clinical study examined the ability of a hyperspectral imaging (HSI) camera (Tivita, Diaspective Vision, Germany) to assess postoperative flap properties in comparison to established clinical parameters. MATERIALS AND METHODS: Thirteen patients with tumor resection and free flap reconstruction were included for camera analysis and another 10 patients as control group. For this purpose, at defined time intervals and under standardized conditions, recordings of transplants 3 to 100 hours postoperatively were performed. Images were used to examine oxygenation (StO 2 %), tissue hemoglobin index, tissue water index, near infrared range perfusion index of free flaps quantitatively and qualitatively. RESULTS: HSI provides values differing between patients observed with no intraindividual significant difference. After 24 hours a mean reduction of 16.77% for StO 2 %, 9.16% for tissue hemoglobin index and 8.46% was observed, going in line with no loss of flap was noted in the observation period. CONCLUSION: HSI is suitable as a noninvasive measure for the evaluation of free flaps in craniomaxillofacial surgery in case of stable imaging conditions with respect to light, surrounding and position of the camera. However, clinical measurements are still the method of choice.

Solid PRF Serves as Basis for Guided Open Wound Healing of the Ridge after Tooth Extraction by Accelerating the Wound Healing Time Course-A Prospective Parallel Arm Randomized Controlled Single Blind Trial.

Systematic evaluations regarding the influence of PRF in ridge sealing are still lacking. To the best of our knowledge, this is the first systemic randomized, controlled, clinical approach dealing with the potential of a systematic applied solid PRF on soft tissue socket healing of molar and premolar extraction sockets with evaluation for up to 90 days. Qualitative and quantitative image analysis showed that PRF contributed to a significantly faster ridge sealing, within the period of 7-10 days in both tooth types. This led to a visibly less contraction at the PRF-treated group sites at day 90. Patients' pain perception demonstrated no statistic significance between both groups (PRF vs. natural healing), but the patients in PRF group seemed to have had less pain throughout the observational period. It becomes evident that PRF is able to serve as a promotor of the secondary wound healing cascade. The guiding capacity of PRF accelerating the process of open ridge healing makes it possible to act as a natural growth factor drug delivery system, providing a more predictable guided open wound healing of the ridge with less contraction of the soft tissue, the latter being a key factor for the subsequent successful dental implantation and oral rehabilitation.

Growth Factor Release within Liquid and Solid PRF.

Aim: The purpose of this study was to obtain data concerning growth factor release within liquid and solid platelet-rich fibrin (PRF) matrices and to estimate the amount of potential interindividual variations as a basis for further preclinical and clinical trials. Therefore, we aimed to determine possible differences in the release of growth factors between liquid and solid PRF. Materials and Methods: Blood samples obtained from four subjects were processed to both liquid and solid PRF matrices using a standard centrifugation protocol. Five growth factors (vascular endothelial growth factor, VEGF; epidermal growth factor, EGF; platelet-derived growth factor-BB, PDGF-BB; transforming growth factor-ß1, TGF-ß1; and matrix metallopeptidase 9, MMP-9) have been evaluated at six time points by ELISA over a total observation period of 10 days (1 h, 7 h, 1 d, 2 d, 7 d, and 10 d). Results: Growth factor release could be measured in all samples at each time point. Comparing liquid and solid PRF matrices, no significant differences were detected (p > 0.05). The mean release of VEGF, TGFß-1, PDGF-BB, and MMP-9 raised to a peak at time point five (day 7) in both liquid and solid PRF matrices. VEGF release was lower in liquid PRF than in solid PRF, whereas those of PDGF-BB and MMP-9 were higher in liquid PRF than in solid PRF at all time points. EGF had its peak release already at time point two after 7 h in liquid and solid matrices (hour 7 EGF solid: mean = 180 pg/mL, SD = 81; EGF liquid: mean = 218 pg/mL, SD = 64), declined rapidly until day 2, and had a second slight peak on day 7 in both groups (day 7 EGF solid: mean = 182 pg/mL, SD = 189; EGF liquid: mean = 81 pg/mL, SD = 70). Conclusions: This study detected growth factor release within liquid and solid PRF matrices with little variations. Further preclinical trials are needed to precisely analyze the growth factor release in larger samples and to better understand their effects on wound healing in different clinical indications.

Comparison of Different Fixation Methods for Combined Histological and Biomolecular Analysis of Fixed and Decalcified Bone Samples.

The combination of histological and biomolecular analyses provides deep understanding of different biological processes and is of high interest for basic and applied research. However, the available analytical methods are still limited, especially when considering bone samples. This study compared different fixation media to identify a sufficient analytical method for the combination of histological, immuno-histological and biomolecular analyses of the same fixed, processed and paraffin embedded bone sample. Bone core biopsies of rats' femurs were fixed in different media (RNAlater + formaldehyde (R + FFPE), methacarn (MFPE) or formaldehyde (FFPE)) for 1 week prior to decalcification by EDTA and further histological processing and paraffin embedding. Snap freezing (unfixed frozen tissue, UFT) and incubation in RNAlater were used as additional controls. After gaining the paraffin sections for histological and immunohistological analysis, the samples were deparaffined and RNA was isolated by a modified TRIZOL protocol. Subsequently, gene expression was evaluated using RT-qPCR. Comparable histo-morphological and immuno-histological results were evident in all paraffin embedded samples of MFPE, FFPE and R + FFPE. The isolated RNA in the group of MFPE showed a high concentration and high purity, which was comparable to the UFT and RNAlater groups. However, in the groups of FFPE and R + FFPE, the RNA quality and quantity were statistically significantly lower when compared to MFPE, UFT and RNAlater. RT-qPCR results showed a comparable outcome in the group of MFPE and UFT, whereas the groups of FFPE and R + FFPE did not result in a correctly amplified gene product. Sample fixation by means of methacarn is of high interest for clinical samples to allow a combination of histological, immunohistological and biomolecular analysis. The implementation of such evaluation method in clinical research may allow a deeper understanding of the processes of bone formation and regeneration.

Extent of Neck Dissection and Cervical Lymph Node Involvement in Oral Squamous Cell Carcinoma.

Introduction: Tumor resection combined with neck dissection (ND) or radiotherapy are established methods for the treatment of patients with oral squamous cell carcinoma (OSCC). However, the extent of ND can lead to postoperative complications. Therefore, for the first time, this study aims to identify lymph node involvement in OSCC performed in a bilateral systematic approach based on oncologic board meetings relying on presurgical magnetic resonance imaging (MRI) and computed tomography (CT). Materials and Methods: In a retrospective single-center study, patients with primary OSCC resection and systematic ND performed in 4 different manners (MRND III bilateral, MRND III left and SND right, MRND III right, SND left, and SND bilateral) were examined. Lymph node involvement allocated to levels was evaluated depending on primary localization and T-stage. Results: A total of 177 consecutive patients (mean age 63.64; 92 female, male 85) were enrolled in this study. A total of 38.98% showed cervical lymph node involvement, and metastases were found in levels 1-4. The distribution of positive lymph node metastases (n=190 LNs) was 39.47% in level 1, 38.95% in level 2, 10.53% in level 3, and 11.05% in level 4. Discussion: In a cohort of OSCC patients with systematic bilateral ND, levels 1 and 2 had positive lymph node involvement, and no lymph node involvement was seen at level 5. Without any clinical or imaging suspicion, ND expanding 5-level MRND should be avoided regardless of the primary tumor localization, T-stage and intraoperative proof of cervical metastases.

Cellular Response of Human Osteoblasts to Different Presentations of Deproteinized Bovine Bone.

Objectives: This study evaluated the cellular response of primary osteoblasts exposed to two different presentations of a low-temperature non-sintered deproteinized bovine bone matrix (DBBM). Materials and methods: Six different baths of a commercially available DBBM block (Bonefill® Porous Block) and one of DBBM granule (Bonefill® Porous) were evaluated to identify the mineral structure and organic or cellular remnants. Samples of the same baths were processed in TRIZOL for RNA extraction and quantification. For the immunologic cell reaction assay, primary human osteoblasts (pOB) were exposed to DBMM block (pOB + B) or granules (pOB + G), or none (control) for 1, 3, or 7 days of cell cultivation. Expression of proinflammatory cytokines by pOB was evaluated by crosslinked ELISA assay. In addition, total DNA amount, as well as cell viability via LDH evaluation, was assessed. Results: Organic remnants were present in DBBM blocks; 45.55% (±7.12) of osteocytes lacunae presented cellular remnants in blocks compared to 17.31% (±1.31) in granules. In three of five batches of blocks, it was possible to isolate bovine RNA. The highest concentration of TGF-ß1 was found in supernatants of pOB + G on day 7 (218.85 ± 234.62 pg/mL) (p < 0.05), whereas pOB + B presented the lowest amount of TGF-ß1 secretion at the end of evaluation (30.22 ± 14.94 pg/mL, p < 0.05). For IL-6 and OPG, there was no statistical difference between groups, while pOB + G induced more IL-8 secretion than the control (3.03 ± 3.38 ng/mL, p < 0.05). Considering the kinetics of cytokine release during the study period, all groups presented a similar pattern of cytokines, estimated as an increasing concentration for IL-6, IL-8, and OPG during cultivation. Adherent cells were observed on both material surfaces on day 7, according to H&E and OPN staining. Conclusion: Neither tested material induced a pronounced inflammatory response upon osteoblast cultivation. However, further studies are needed to elucidate the potential influence of organic remnants in bone substitute materials on the regeneration process.

A Standardized g-Force Allows the Preparation of Similar Platelet-Rich Fibrin Qualities Regardless of Rotor Angle.

Platelet-rich fibrin (PRF) is an autologous blood concentrate that supports tissue regeneration. The effect of the centrifuge rotor angle in the fabrication of PRF is still not fully elucidated. The hypothesis of this study is: When applying the same g-force (relative centrifugal force [RCF]) and centrifugation time, PRF components and bioactivity are not modified using either a swing-out rotor or a fixed angle rotor. For this purpose, peripheral blood samples (from five donors) were used to gain solid (710 ×g, 8 min) and liquid (44 ×g, 8 min) PRF matrices using three different centrifuges (one fixed angle as a control and two different swing-out rotor centrifuges). The physical characteristics of the solid PRF were measured to evaluate the clot formation and cellular distribution. The liquid PRF was used to evaluate the cell number, bioactivity, and influence on primary human osteoblasts (pOBs) and primary human fibroblasts (pHFs) in vitro. Solid PRF clots were significantly larger in the group of fixed rotor centrifuges compared with either of the two evaluated swing-out rotor centrifuges. No differences were observed when evaluating the cellular distribution within the solid PRF. No statistically significant differences were documented in the cell's density in liquid PRF samples (platelets, lymphocytes, neutrophils, eosinophils, and basophils) among the differently gained PRF samples. No statistically significant differences were documented for the released growth factors (vascular endothelial growth factor, epidermal growth factor, and transforming growth factor beta 1) over 7 days. pOBs and pHFs viability after treatment with PRF conditioned media showed no statistically significant differences between the evaluated groups. However, the number of adherent cells treated with PRF obtained with the use of the fixed angle rotor was significantly higher when compared with those treated with PRF obtained by using the swing-out rotors. The presented results confirm that regardless of the centrifuge rotor used, the components and bioactivity of solid and liquid PRF matrices are modified by the applied RCF and centrifugation time. These findings are of great importance for highlighting the essential role of adapting the centrifugation protocols when using different centrifuges and to correctly report the used centrifugation protocols in scientific research to allow for reproducible results. Impact statement Platelet-rich fibrin (PRF) is prepared from autologous peripheral blood and is widely applied in research and clinical treatments. The centrifugation parameters used during the preparation of PRF directly influence its components and bioactivity. By using a standardized protocol, the present study demonstrated that adapting various centrifuges to a standardized relative centrifugal force and centrifugation protocol resulted in reproducible PRF matrices with similar bioactivity, regardless of the centrifuge rotor angle. These findings underline the necessity to carefully adapt and correctly report the used centrifuge and centrifugation protocols in scientific research to allow reproducible results.

Covalent linkage of sulfated hyaluronan to the collagen scaffold Mucograft® enhances scaffold stability and reduces proinflammatory macrophage activation in vivo.

Sulfated glycosaminoglycans (sGAG) show interaction with biological mediator proteins. Although collagen-based biomaterials are widely used in clinics, their combination with high-sulfated hyaluronan (sHA3) is unexplored. This study aims to functionalize a collagen-based scaffold (Mucograft®) with sHA3 via electrostatic (sHA3/PBS) or covalent binding to collagen fibrils (sHA3+EDC/NHS). Crosslinking without sHA3 was used as a control (EDC/NHS Ctrl). The properties of the sHA3-functionalized materials were characterized. In vitro growth factor and cytokine release after culturing with liquid platelet-rich fibrin was performed by means of ELISA. The cellular reaction to the biomaterials was analyzed in a subcutaneous rat model. The study revealed that covalent linking of sHA3 to collagen allowed only a marginal release of sHA3 over 28 days in contrast to electrostatically bound sHA3. sHA3+EDC/NHS scaffolds showed reduced vascular endothelial growth factor (VEGF), transforming growth factor beta 1 (TGF-ß1) and enhanced interleukin-8 (IL-8) and epithelial growth factor (EGF) release in vitro compared to the other scaffolds. Both sHA3/PBS and EDC/NHS Ctrl scaffolds showed a high proinflammatory reaction (M1: CD-68+/CCR7+) and induced multinucleated giant cell (MNGC) formation in vivo. Only sHA3+EDC/NHS scaffolds reduced the proinflammatory macrophage M1 response and did not induce MNGC formation during the 30 days. SHA3+EDC/NHS scaffolds had a stable structure in vivo and showed sufficient integration into the implantation region after 30 days, whereas EDC/NHS Ctrl scaffolds underwent marked disintegration and lost their initial structure. In summary, functionalized collagen (sHA3+EDC/NHS) modulates the inflammatory response and is a promising biomaterial as a stable scaffold for full-thickness skin regeneration in the future.

Efficacy of platelet-rich fibrin in promoting the healing of extraction sockets: a systematic review.

PURPOSE: To address the focused question: in patients with freshly extracted teeth, what is the efficacy of platelet-rich fibrin (PRF) in the prevention of pain and the regeneration of soft tissue and bone compared to the respective control without PRF treatment? METHODS: After an electronic data search in PubMed database, the Web of Knowledge of Thomson Reuters and hand search in the relevant journals, a total of 20 randomized and/or controlled studies were included. RESULTS: 66.6% of the studies showed that PRF significantly reduced the postoperative pain, especially in the first 1-3 days after tooth extraction. Soft tissue healing was significantly improved in the group of PRF compared to the spontaneous wound healing after 1 week (75% of the evaluated studies). Dimensional bone loss was significantly lower in the PRF group compared to the spontaneous wound healing after 8-15 weeks but not after 6 months. Socket fill was in 85% of the studies significantly higher in the PRF group compared to the spontaneous wound healing. CONCLUSIONS: Based on the analyzed studies, PRF is most effective in the early healing period of 2-3 months after tooth extraction. A longer healing period may not provide any benefits. The currently available data do not allow any statement regarding the long-term implant success in sockets treated with PRF or its combination with biomaterials. Due to the heterogeneity of the evaluated data no meta-analysis was performed.

Multinucleated Giant Cells Induced by a Silk Fibroin Construct Express Proinflammatory Agents: An Immunohistological Study.

Multinucleated giant cells (MNGCs) are frequently observed in the implantation areas of different biomaterials. The main aim of the present study was to analyze the long-term polarization pattern of the pro- and anti-inflammatory phenotypes of macrophages and MNGCs for 180 days to better understand their role in the success or failure of biomaterials. For this purpose, silk fibroin (SF) was implanted in a subcutaneous implantation model of Wistar rats as a model for biomaterial-induced MNGCs. A sham operation was used as a control for physiological wound healing. The expression of different inflammatory markers (proinflammatory M1: CCR-7, iNos; anti-inflammatory M2: CD-206, CD-163) and tartrate-resistant acid phosphatase (TRAP) and CD-68 were identified using immunohistochemical staining. The results showed significantly higher numbers of macrophages and MNGCs within the implantation bed of SF-expressed M1 markers, compared to M2 markers. Interestingly, the expression of proinflammatory markers was sustained over the long observation period of 180 days. By contrast, the control group showed a peak of M1 macrophages only on day 3. Thereafter, the inflammatory pattern shifted to M2 macrophages. No MNGCs were observed in the control group. To the best of our knowledge, this is study is the first to outline the persistence of pro-inflammatory MNGCs within the implantation bed of SF and to describe their long-term kinetics over 180 days. Clinically, these results are highly relevant to understand the role of biomaterial-induced MNGCs in the long term. These findings suggest that tailored physicochemical properties may be a key to avoiding extensive inflammatory reactions and achieving clinical success. Therefore, further research is needed to elucidate the correlation between proinflammatory MNGCs and the physicochemical characteristics of the implanted biomaterial.

Biologization of Pcl-Mesh Using Platelet Rich Fibrin (Prf) Enhances Its Regenerative Potential In Vitro.

INTRODUCTION: Resorbable synthetic scaffolds are promising for different indications, especially in the context of bone regeneration. However, they require additional biological components to enhance their osteogenic potential. In addition to different cell types, autologous blood-derived matrices offer many advantages to enhance the regenerative capacity of biomaterials. The present study aimed to analyze whether biologization of a PCL-mesh coated using differently centrifuged Platelet rich fibrin (PRF) matrices will have a positive influence on primary human osteoblasts activity in vitro. A polymeric resorbable scaffold (Osteomesh, OsteoporeTM (OP), Singapore) was combined with differently centrifuged PRF matrices to evaluate the additional influence of this biologization concept on bone regeneration in vitro. Peripheral blood of three healthy donors was used to gain PRF matrices centrifuged either at High (710× g, 8 min) or Low (44× g, 8 min) relative centrifugal force (RCF) according to the low speed centrifugation concept (LSCC). OP-PRF constructs were cultured with pOBs. POBs cultured on the uncoated OP served as a control. After three and seven days of cultivation, cell culture supernatants were collected to analyze the pOBs activity by determining the concentrations of VEGF, TGF-ß1, PDGF, OPG, IL-8, and ALP- activity. Immunofluorescence staining was used to evaluate the Osteopontin expression of pOBs. After three days, the group of OP+PRFLow+pOBs showed significantly higher expression of IL-8, TGF-ß1, PDGF, and VEGF compared to the group of OP+PRFHigh+pOBs and OP+pOBs. Similar results were observed on day 7. Moreover, OP+PRFLow+pOBs exhibited significantly higher activity of ALP compared to OP+PRFHigh+pOBs and OP+pOBs. Immunofluorescence staining showed a higher number of pOBs adherent to OP+PRFLow+pOBs compared to the groups OP+PRFHigh+pOBs and OP+pOBs. To the best of our knowledge, this study is the first to investigate the osteoblasts activity when cultured on a PRF-coated PCL-mesh in vitro. The presented results suggest that PRFLow centrifuged according to LSCC exhibits autologous blood cells and growth factors, seem to have a significant effect on osteogenesis. Thereby, the combination of OP with PRFLow showed promising results to support bone regeneration. Further in vivo studies are required to verify the results and carry out potential results for clinical translation.

Genotoxic and cytotoxic potential of methacrylate-based orthodontic adhesives.

OBJECTIVES: The biocompatibility of methacrylate-based adhesives is a topic that is intensively discussed in dentistry. Since only limited evidence concerning the cyto- and genotoxicity of orthodontic adhesives is available, the aim of this study was to measure the genotoxic potential of seven orthodontic methacrylate-based adhesives. MATERIALS AND METHODS: The XTT assay was utilized to determine the cytotoxicity of Assure Plus, Assure Bonding Resin, ExciTE F, OptiBond Solo Plus, Scotchbond Universal Adhesive, Transbond MIP, and Transbond XT after an incubation period of 24 h on human gingival fibroblasts. We also performed the γH2AX assay to explore the genotoxic potential of the adhesives within cytotoxic dose ranges after an incubation period of 6 h. RESULTS: The XTT assay showed a concentration-dependent reduction in cell viability. The decrease in cellular viability was in the same dose range most significant for Assure Plus, rendering it the adhesive material with the highest cytotoxicity. Employing the γH2AX assay, a concentration-dependent increase in H2AX phosphorylation was detected, indicating induction of DNA damage. CONCLUSIONS: For most products, a linear correlation between the material concentration and γH2AX foci was observed. The most severe effect on γH2AX focus induction was found for Transbond MIP, which was the only adhesive in the test group containing the co-initiator diphenyliodonium hexafluorophosphate (DPIHP). CLINICAL RELEVANCE: The data indicate that orthodontic adhesives, notably Transbond MIP, bear a genotoxic potential. Since the study was performed with in vitro cultivated cells, a direct translation of the findings to in vivo exposure conditions should be considered with great diligence.

Effects of rotor angle and time after centrifugation on the biological in vitro properties of platelet rich fibrin membranes.

This study evaluated the impact of rotor angle and time of storage after centrifugation on the in vitro biological properties of platelet-rich fibrin (PRF) membranes. Blood samples (n = 9) were processed with a vertical fixed-angle (V) or a swing-out horizontal (H) centrifuge, with 20-60 min of sample storage after centrifugation. Leukocytes, platelets, and red blood cells were counted, and fibrin architecture was observed by scanning electron microscopy (SEM). The release of FGF2, PDGFbb, VEGF, IL-6, and IL-1ß was measured after incubation on culture media for 7-21 days. Cell content was equivalent in all experimental groups (p > .05). The fibrin matrix was similar for fixed-angle and horizontal centrifugation. Horizontal centrifugation induced a twofold increase in PDGF and 1.7× increase on FGF release as compared to V samples, while IL-1ß was significantly reduced (p < .05). No significant difference was observed on the release of growth factors and cytokines at different times after centrifugation (p < .05). These data suggest that both angles of centrifugation produce PRF membranes with similar structure and cellularity, but horizontal centrifugation induces a higher release of growth factors. Higher times of storage after centrifugation did not impact on cell content and the release of growth factors.

Multinucleated giant cells within the in vivo implantation bed of a collagen-based biomaterial determine its degradation pattern.

OBJECTIVES: The aim of the present study was to characterize the cellular reaction to a xenogeneic resorbable collagen membrane of porcine origin using a subcutaneous implantation model in Wistar rats over 30 days. MATERIALS AND METHODS: Ex vivo, liquid platelet-rich fibrin (PRF), a leukocyte and platelet-rich cell suspension, was used to evaluate the blood cell membrane interaction. The material was implanted subcutaneously in rats. Sham-operated rats without biomaterial displayed physiological wound healing (control group). Histological, immunohistological, and histomorphometric analyses were focused on the inflammatory pattern, vascularization rate, and degradation pattern. RESULTS: The membrane induced a large number of mononuclear cells over the observation period, including lymphocytes, macrophages, and fibroblasts. After 15 days, multinucleated giant cells (MNGCs) were observed on the biomaterial surface. Their number increased significantly, and they proceeded to the center of the biomaterial on day 30. These cells highly expressed CD-68, calcitonin receptor, and MMP-9, but not TRAP or integrin-ß3. Thus, the membrane lost its integrity and underwent disintegration as a consequence of the induction of MNGCs. The significant increase in MNGC number correlated with a high rate of vascularization, which was significantly higher than the control group. Physiological wound healing in the control group did not induce any MNGCs at any time point. Ex vivo blood cells from liquid-PRF did not penetrate the membrane. CONCLUSION: The present study suggests a potential role for MNGCs in biomaterial degradation and questions whether it is beneficial to accept them in clinically approved biomaterials or focus on biomaterials that induce only mononuclear cells. Thus, further studies are necessary to identify the function of biomaterial-induced MNGCs. CLINICAL RELEVANCE: Understanding the cellular reaction to biomaterials is essential to assess their suitability for specific clinical indications and outline the potential benefit of specific group of biomaterials in the respective clinical indications.

Thermal treatment at 500 °C significantly reduces the reaction to irregular tricalcium phosphate granules as foreign bodies: An in vivo study.

Evaporation of phosphate species during thermal treatment (> 400 °C) of calcium phosphates leads to the formation of an alkaline layer on their surface. The aim of this study was to evaluate the hypothesis that the biological response of thermally treated calcium phosphates is modified by the presence of such an alkaline layer on their surface. For this purpose, 0.125-0.180 mm α- and ß-tricalcium phosphate (TCP) granules were obtained by crushing and size classification, with some being subjected to thermal treatment at 500 °C. The four types of granules (α-TCP, ß-TCP, α-TCP-500 °C, and ß-TCP-500 °C) were implanted subcutaneously and orthotopically in rats. Sham operations served as control. Subcutaneously, α-TCP and ß-TCP induced significantly more multinucleated giant cells (MNGCs) than calcined granules. Most of the induced MNGCs were TRAP-negative, CD-68 positive and cathepsin K-negative, reflecting a typical indication of a reaction with a foreign body. The vessel density was significantly higher in the α-TCP and ß-TCP groups than it was in the α-TCP-500 °C and ß-TCP-500 °C groups. In the femur model, ß-TCP-500 °C induced significantly more new bone formation than that induced by ß-TCP. The granule size was also significantly larger in the ß-TCP-500 °C group, making it more resistant to degradation than ß-TCP. The MNGC density was higher in the α-TCP and ß-TCP groups than in the α-TCP-500 °C and ß-TCP-500 °C groups, including cathepsin-positive, CD-68 positive, TRAP-positive and TRAP-negative MNGCs. In conclusion, this study confirms that the biological response of calcium phosphates was affected by the presence of an alkaline layer on their surface. Thermally-treated α-TCP and ß-TCP granules produced significantly fewer MNGCs and were significantly less degraded than non-thermally-treated α-TCP and ß-TCP granules. Thermally treating α-TCP and ß-TCP granules shifts the reaction from a foreign body reaction towards a physiological reaction by downregulating the number of induced MNGCs and enhancing degradation resistance.