In Vitro and Ex Vivo Kinetic Release Profile of Growth Factors and Cytokines from Leucocyte- and Platelet-Rich Fibrin (L-PRF) Preparations.

L-PRF is an autologous blood-derived biomaterial (ABDB) capable of releasing biologically active agents to promote healing. Little is known about its release profile of growth factors (GFs), cytokines, and MMPs. This study reported the in vitro and ex vivo release kinetics of GFs, cytokines, and MMPs from L-PRF at 6, 24, 72, and 168 h. The in vitro release rates of PDGF, TGF-ß1, EGF, FGF-2, VEGF, and MMPs decreased over time with different rates, while those of IL-1ß, IL-6, TNF-α, IL-8, and IL-10 were low at 6 h and then increased rapidly for up to 24 h and subsequently decreased. Of note, the release rates of the GFs followed first-order kinetics both in vitro and ex vivo. Higher rates of release were found ex vivo, suggesting that significant amounts of GFs were produced by the local cells within the wound. In addition, the half-life times of GFs locally produced in the wound, including PDGF-AA, PDGF-AB/BB, and VEGF, were significantly extended (p < 0.05). This work demonstrates that L-PRF can sustain the release of GFs and cytokines for up to 7 days, and it shows that the former can activate cells to produce additional mediators and amplify the communication network for optimizing the wound environment, thereby enhancing healing.

Increased local concentrations of growth factors from leucocyte- and platelet-rich fibrin do not translate into improved alveolar ridge preservation: An intra-individual mechanistic randomized controlled trial.

AIM: Leucocyte- and platelet-rich fibrin (L-PRF) has been tested for enhancing alveolar ridge preservation (ARP), but little is known about the local release profile of growth factors (GF), and the clinical equipoise related to its efficacy remains. This study compared the patterns of GF release, early soft tissue healing, and alveolar ridge resorption following unassisted healing and L-PRF application in non-molar extraction sockets. MATERIALS AND METHODS: Atraumatic tooth extraction of two hopeless teeth per patient was followed by unassisted healing or L-PRF placement to fill the socket in 18 systemically healthy, non-smoking subjects. This intra-individual trial was powered to assess changes in horizontal alveolar ridge dimensions 1 mm below the crest of alveolar bone. GF concentrations in wound fluid were assessed with a multiplex assay at 6, 24, 72, and 168 h. Early healing was evaluated with the wound healing index and changes in soft tissue volumes on serial digital scans. Hard tissue changes were measured on superimposed CBCT images after 5 months of healing. RESULTS: L-PRF resulted in higher GF concentrations in wound fluid (WF) than in the control, but no differences in release patterns or time of peak were observed. No inter-group differences in early healing parameters were observed. Alveolar bone resorption was observed in both groups. No significant inter-group differences were observed in hard tissue healing 1, 3, or 5 mm apical to the original bone crest or in the ability to digitally plan a prosthetically guided implant with or without bone augmentation. CONCLUSIONS: L-PRF increased the GF concentrations in WF of extraction sockets without shifting the pattern observed in unassisted healing, while the increased delivery did not translate into clinical benefits in early wound healing or ARP. The current findings question the assumption that increased local concentrations of GF by L-PRF translate into improved clinical outcomes. Additional definitive studies are needed to establish the benefits of L-PRF in ARP (ClinicalTrials.gov NCT03985033).

Transcription factor 7-like 2-associated signaling mechanism in regulating cementum generation by the NF-κB pathway.

Cementum regeneration is an important and challenging stage in periodontal tissue engineering and regeneration. Pathosis of the periodontium, including cementum, is important in precision diagnosis and obstinate treatment of systemic diseases, such as diabetes, leukemia, and Acquired Immune Deficiency Syndrome. Here, we found that during periodontium development, transcription factor 7-like 2 (Tcf7l2) was widely expressed in the periodontium and dental sac. In mouse cementoblast cell line (OCCM-30), the activation of NF-κB and cementoblast mineralization was significantly reduced when Tcf7l2 gene was silenced. Moreover, Tcf7l2 has a positive effect on NF-κB and cementoblast mineralization. Therefore, Tcf7l2 promotes cementum formation through the NF-κB pathway. In addition, we found a decreased expression of phosphorylated p65 and a thin layer of cementum in Tcf7l2fl/fl mice. These results suggest that Tcf7l2, which accelerates cementum formation by activating NF-κB, has great potential in the treatment of periodontitis and provide guidance for periodontal tissue regeneration.

Fluid platelet-rich fibrin stimulates greater dermal skin fibroblast cell migration, proliferation, and collagen synthesis when compared to platelet-rich plasma.

BACKGROUND: Regenerative therapies in the field of facial aesthetics have become a growing field of interest with many recent advancements made over the past decade to meet the growing worldwide demand. While first versions of platelet-derived concentrates were formulated with anticoagulants (PRP), recent modifications to centrifugation speeds and times have permitted the development of a liquid platelet-rich fibrin (fluid-PRF) without use of anticoagulants. OBJECTIVE: To compare this entirely natural platelet concentrate (fluid-PRF) to formally utilized PRP on skin cell behavior and regeneration. METHODS: Dermal skin fibroblast was cultivated with either fluid-PRF or PRP and investigated for their ability to promote/influence cell viability, migration, spreading, proliferation, and mRNA levels of known mediators of dermal biology including PDGF, TGF-beta, and fibronectin. RESULTS: All platelet concentrates were nontoxic to cells demonstrating high cell survival. Skin fibroblasts migrated over 350% more in fluid-PRF when compared to control and PRP (200% increase). Fluid-PRF also significantly induced greater cell proliferation at 5 days. While both PRP and fluid-PRF induced significantly elevated cell mRNA levels of PDGF, it was observed that TGF-beta, collagen 1, and fibronectin mRNA levels were all significantly highest in the fluid-PRF group. Lastly, fluid-PRF demonstrated a significantly greater ability to induce collagen matrix synthesis when compared to PRP. CONCLUSION: The findings from the present study demonstrate greater regenerative potential of fluid-PRF on human skin fibroblasts. Future clinical use of fluid-PRF in the field of facial aesthetics is necessary to further evaluate the potential advantages of anticoagulant removal from platelet concentrates.

The interactions of dendritic cells with osteoblasts on titanium surfaces: an in vitro investigation.

OBJECTIVES: Osteoimmune interactions possess a critical part in the integration of materials and hosts. Dendritic cells (DCs) are the most common members of osteoimmune cells family. The titanium surfaces of dental implants tend to promote a mature dendritic cell phenotype with increased proinflammatory secretion. However, very little is known to the effects of this microenvironment on the behaviors of cells around implants, especially osteoblasts, and how the tissue integrations take place on such biomaterial surfaces. Therefore, the present study was to investigate the interactions of DCs with osteoblasts on titanium surfaces. DCs seeded on PT and SLA titanium surfaces were compared by assays for the proliferations, surface markers, and inflammatory genes expressions. MATERIALS AND METHODS: DCs seeded on PT and SLA titanium surfaces were compared by assays for the proliferations, surface markers, and inflammatory genes expressions. Next, we harvested the dendritic cell-conditioned medium (CM) and investigated the effects of CM on MC3T3-E1. RESULTS: The results showed an increase in CD86 and proinflammatory expressions of DCs seeded on PT and SLA surfaces, as well as a decrease in anti-inflammatory cytokines. The CM from titanium surfaces inhibited the osteoblast differentiation with reduced expression of osteogenic genes RUNX2, COL1, ALP, and OCN and decreased ALP activity as well as Alizarin red staining. CONCLUSION: These findings suggested that titanium surfaces switch DCs toward maturation phenotypes and thus inhibit the differentiation and mineralization of osteoblasts. CLINICAL RELEVANCE: Knowing the significant impact of immune cells on osteogenesis behaviors, some efforts to decrease the immune reaction might be of clinical significance. Favorable immune environments can increase the dental implants survival rate in patients.

Injectable-platelet rich fibrin using the low speed centrifugation concept improves cartilage regeneration when compared to platelet-rich plasma.

The aim of the present study was to evaluate the effect of injectable platelet-rich fibrin (i-PRF) on cultivated chondrocytes and osteochondral regeneration in critical-sized osteochondral defect of the rabbit's knee in comparison to autologous platelet-rich plasma (PRP). Chondrocytes were first investigated for their ability to proliferate and differentiate in response to PRP and i-PRF. Thereafter, full-thickness critical-sized osteochondral defects 5 mm in diameter and 5 mm in depth were created in the knee joint of 12 adult female New Zealand White rabbits. Defects were regenerated with either PRP or i-PRF and compared to control. Animals were sacrificed at 4 and 12 weeks postoperatively and evaluated histologically by macroscopic and microscopic examination for cartilage regeneration. i-PRF significantly promoted chondrocyte proliferation and mRNA levels of Sox9, collagen type II, and aggrecan when compared to PRP and control. Histological analysis revealed that at 4 weeks, macroscopic ICRS scores from the i-PRF group were significantly enhanced when compared to the PRP and control groups. At 12 weeks post surgery, the microscopic ICRS scores demonstrated that the i-PRF group significantly improved cartilage regeneration when compared to PRP. In conclusion, the use of i-PRF using the low speed centrifugation concept significantly promoted chondrocyte activity and further improved cartilage regeneration when compared to PRP. The histological results revealed early and better cartilage regeneration within 4 weeks postoperatively when i-PRF was utilized and the results were maintained at 12 weeks. Future clinical studies are now needed investigating the regenerative potential of i-PRF in comparison to PRP for knee regeneration.

Near-infrared light-triggered drug delivery system based on black phosphorus for in vivo bone regeneration.

A near-infrared (NIR) light-triggered drug delivery platform is produced by incorporating SrCl2 and BP nanosheets (BPs) into poly(lactic-co-glycolic acid) (PLGA) for bone regeneration. The fabricated BP-SrCl2/PLGA microspheres show efficient NIR absorption and photothermal effects due to the BPs. The NIR-triggered release behavior of Sr2+ by flawing the PLGA shells is investigated and the microspheres exhibit excellent cell viability and biodegradability. Implantation of the BP-SrCl2/PLGA microspheres into a rat femoral defect demonstrates good tissue compatibility and excellent bone regeneration capacity under NIR light irradiation. Our study indicates that local release of Sr2+ at optimal time periods controlled by NIR irradiation improves bone regeneration significantly and this NIR-triggered drug delivery system composed of BPs is suitable for therapies requiring precise control at specific time.

The role of macrophage polarization on fibroblast behavior-an in vitro investigation on titanium surfaces.

OBJECTIVES: This study investigated the effect of smooth and rough titanium surface topographies on macrophage polarization and their influence on gingival fibroblast behavior cultured on titanium surfaces. MATERIALS AND METHODS: RAW 264.7 macrophages were seeded on smooth (pickled titanium (PT)) and rough Sand-blasted with Large grit particles followed by Acid-etching (SLA) titanium surfaces and first investigated for macrophage polarization towards tissue-inflammatory M1 macrophages or wound-healing M2 macrophages. Thereafter, culture media collected from macrophages on both surfaces were cultured with gingival fibroblasts seeded on their respective topographies. All experiments were performed in triplicate with three independent experiments. RESULTS: Macrophages seeded on SLA surfaces polarized towards tissue-inflammatory M1 macrophages at early time points. Immunofluorescent staining and RT-PCR analysis demonstrated higher levels of iNOS and gene expression of IL-1ß, IL-6, and TNF-alpha on SLA surfaces at 3 days when compared to both tissue culture plastic (TCP) and PT surfaces (p < 0.001). Very little differences were found between smooth PT surfaces and TCP. Interestingly, proliferation assay (CCK-8) suggested that conditioned media (CM) from macrophages seeded on SLA surfaces drastically inhibited gingival fibroblast proliferation at 3 and 5 days (p < 0.001). Meanwhile, CM from macrophages cultured on SLA surfaces also significantly reduced collagen 1 synthesis on SLA surfaces at 14 days as assessed by immunofluorescent staining (p < 0.001). CONCLUSION: The results from this study demonstrate that the polarization of macrophages towards a pro-inflammatory (M1) phenotype on SLA surfaces may have a negative impact on gingival fibroblast behavior on titanium surfaces. Future strategies to better modulate macrophage polarization should be investigated to support a favorable immune response and encourage tissue integration. CLINICAL RELEVANCE: As SLA surfaces have a potential to shift macrophages towards tissue-inflammatory M1 macrophages, this might be a negative impact for soft tissue healing. Therefore, SLA surfaces should be kept within the bone, as when in contact with soft tissue, they are prone to support a lack of soft tissue integration leading to inflammation.

Effects of an injectable platelet-rich fibrin on osteoblast behavior and bone tissue formation in comparison to platelet-rich plasma.

Platelet-rich plasma (PRP) has been utilized for many years as a regenerative agent capable of inducing vascularization of various tissues using blood-derived growth factors. Despite this, drawbacks mostly related to the additional use of anti-coagulants found in PRP have been shown to inhibit the wound healing process. For these reasons, a novel platelet concentrate has recently been developed with no additives by utilizing lower centrifugation speeds. The purpose of this study was therefore to investigate osteoblast behavior of this novel therapy (injectable-platelet-rich fibrin; i-PRF, 100% natural with no additives) when compared to traditional PRP. Human primary osteoblasts were cultured with either i-PRF or PRP and compared to control tissue culture plastic. A live/dead assay, migration assay as well as a cell adhesion/proliferation assay were investigated. Furthermore, osteoblast differentiation was assessed by alkaline phosphatase (ALP), alizarin red and osteocalcin staining, as well as real-time PCR for genes encoding Runx2, ALP, collagen1 and osteocalcin. The results showed that all cells had high survival rates throughout the entire study period irrespective of culture-conditions. While PRP induced a significant 2-fold increase in osteoblast migration, i-PRF demonstrated a 3-fold increase in migration when compared to control tissue-culture plastic and PRP. While no differences were observed for cell attachment, i-PRF induced a significantly higher proliferation rate at three and five days when compared to PRP. Furthermore, i-PRF induced significantly greater ALP staining at 7 days and alizarin red staining at 14 days. A significant increase in mRNA levels of ALP, Runx2 and osteocalcin, as well as immunofluorescent staining of osteocalcin was also observed in the i-PRF group when compared to PRP. In conclusion, the results from the present study favored the use of the naturally-formulated i-PRF when compared to traditional PRP with anti-coagulants. Further investigation into the direct role of fibrin and leukocytes contained within i-PRF are therefore warranted to better elucidate their positive role in i-PRF on tissue wound healing.

Behavior of Gingival Fibroblasts on Titanium Implant Surfaces in Combination with either Injectable-PRF or PRP.

Various strategies have been employed to speed tissue regeneration using bioactive molecules. Interestingly, platelet concentrates derived from a patient's own blood have been utilized as a regenerative strategy in recent years. In the present study, a novel liquid platelet formulation prepared without the use of anti-coagulants (injectable-platelet-rich fibrin, i-PRF) was compared to standard platelet-rich plasma (PRP) with gingival fibroblasts cultured on smooth and roughened titanium implant surfaces. Standard PRP and i-PRF (centrifuged at 700 rpm (60× g) for 3 min) were compared by assays for fibroblast biocompatibility, migration, adhesion, proliferation, as well as expression of platelet-derived growth factor (PDGF), transforming growth factor-ß (TGF-ß), collagen1 (COL1) and fibronectin (FN). The results demonstrate that i-PRF induced significantly higher cell migration, as well as higher messenger RNA (mRNA) levels of PDGF, TGF-ß, collagen1 and fibronectin when compared to PRP. Furthermore, collagen1 synthesis was highest in the i-PRF group. These findings demonstrate that liquid platelet concentrates can be formulated without the use of anticoagulants and present much translational potential for future research. Future animal and clinical trials are now necessary to further investigate the potential of utilizing i-PRF for soft tissue regenerative protocols in combination with various biomaterials.

Tests of a practical visible-NIR imaging Fourier transform spectrometer for biological and chemical fluorescence emission measurements.

An imaging Fourier transform spectrometer (IFTS) designed for fluorescence emission measurements is reported. The spectral range extension from NIR to visible of the system is realized by using a simple and low-cost optical beam-folding position-tracking technique. Spectral resolution as high as 9.78cm(-1)(0.4nm at 632.8nm) and maximum image resolution up to 300x300 pixels are proved by the system tests on its optical performances. Imaging fluorescence spectra acquisition of quantum dot clusters and single 200nm diameter fluorescent beads have demonstrated the system's potential for high throughput imaging spectroscopic measurements of fluorescent biological and chemical samples.

Near UV-near IR Fourier transform spectrometer using the beam-folding position-tracking method based on retroreflectors.

A near UV-near IR Fourier transform spectrometer based on a beam-folding position-tracking method realized by using retroreflectors is reported. The use of retroreflectors maintains all beams in the beam-fold arrangement in parallel with the incident beams. The beam-folding interferometer used for position tracking is arranged to have optical path symmetry with the measurement interferometer in the zero path difference position of the measurement interferometer, and the vertex of the movable retroreflector in the measurement interferometer is arranged very close to the midpoint of the vertices of two movable retroreflectors in the position-tracking interferometer. These measures keep the equivalent optical axis of the position-tracking interferometer well in line with that of the measurement interferometer even with translational misalignments. Therefore, the change in the optical path difference of the position-tracking interferometer is always synchronous to that of the measurement interferometer during the scanning process. That is, the position-tracking error can be suppressed to very small values during a scan. We have demonstrated a UV-near IR Fourier transform spectrometer with a standard quality ball-bearing translation stage achieving a resolution close to the theoretical resolution of approximately 0.28 cm(-1) at the He-Ne laser wavelength when the scan distance reaches the travel distance of over 2 cm. This was achieved without the need for elaborate optics, sophisticated detecting electronics, and high-precision servomotion control.

Fourier transform ultraviolet-visible spectrometer based on a beam-folding technique.

A beam-folding technique in optical interferometry, where the number of beam folds used can be very large, is reported. This technique can be used as a low-cost position-tracking method in a Fourier transform spectrometer (FTS) to cover the broad spectral range from UV to IR. The main advantage gained is the simple position-tracking algorithm used in sampling the interferogram. We have developed a UV-visible FTS, whose wavelength coverage is limited only by the optical elements (350 nm(-1) microm with off-the-shelf components). Preliminary results show that it can achieve a resolution of approximately 4 cm(-1) even with a ball-bearing translation stage.

Underwater cytometer for in situ measurement of marine phytoplankton by a technique combining laser-induced fluorescence and laser Doppler velocimetry.

We present a new type of flow cytometer that can operate underwater for a long time, as long as days, for measuring the size distribution, concentration, and biomass of marine phytoplankton. The major improvement of the instrument over existing techniques is the elimination of sample preparation, which is achieved with a laser Doppler crossed-beam arrangement for both defining a measurement volume and measuring the speed of the particle traversing it. By simultaneously sampling the laser-induced fluorescence signal and the Doppler signals, the technique can discriminate sizes of phytoplankton.