Activation of cell adhesion and migration is an early event of platelet-rich plasma (PRP)-dependent stimulation of human adipose-derived stem/stromal cells.

Stem cell therapy is a promising treatment in regenerative medicine. Human adipose-derived stem/stromal cells (hASCs), a type of mesenchymal stem cell, are easy to harvest. In plastic and aesthetic surgery, hASC may be applied in the treatment of fat grafting, wound healing, and scar remodeling. Platelet-rich plasma (PRP) contains various growth factors, including platelet-derived growth factor (PDGF), which accelerates wound healing. We previously reported that PRP promotes the proliferation of hASC via multiple signaling pathways, and we evaluated the effect of PRP on the stimulation of hASC adhesion and migration, leading to the proliferation of these cells. When hASCs were treated with PRP, AKT, ERK1/2, paxillin and RhoA were rapidly activated. PRP treatment led to the formation of F-actin stress fibers. Strong signals for integrin ß1, paxillin and RhoA at the cell periphery of RPR-treated cells indicated focal adhesion. PRP promoted cell adhesion and movement of hASC, compared with the control group. Imatinib, an inhibitor of the PDGF receptor tyrosine kinase, inhibited the promotion of PRP-dependent cell migration. PDGF treatment of hASCs also stimulated cell adhesion and migration but to a lesser extent than PRP treatment. PRP promoted the adhesion and the migration of hASC, mediated by the activation of AKT in the integrin signaling pathway. PRP treatment was more effective than PDGF treatment in enhancing cell migration. Thus, the ability of PRPs to promote migration of hASC to enhance cell growth is evident.

A roadmap for safety during facial filler injections: A fresh frozen cadaver study.

BACKGROUND: Filler injections are commonly applied to reshape facial contouring. However, cadaveric injections of filler for facial contouring on the whole face, followed by anatomic analysis and measurement, have rarely been reported. This study aimed to provide comprehensive anatomical information, including topographies and roadmap of injection point entry, penetration depth, filler location, the hierarchy of facial structure, and vital vascular course. METHODS: Thirty faces on fresh frozen cadaver heads were used for this anatomic study. The whole face was divided into seven facial zones and 14 injection points for penetration depth measurement and cadaveric injection. Static periosteum injections with a sharp-needle technique were performed. Specimens were then dissected to observe the precise locations of fillers and their relationships with surrounding anatomic structures. RESULTS: The topography of penetration depth gradually increased from the upper face to the middle face, lower face, and temporal region. Most of the injected hyaluronic acid filler flowed backward to the loose areolar tissue layer between the superficial musculoaponeurotic system and periosteum or deep fascia. Multilevel layer distributions and anastomosis of the vessels were found in the face, especially in the glabella, dorsum nasi, and temporal regions. CONCLUSIONS: This study can provide clinicians with a comprehensive reference for facial contouring injections: topographies of the injection point and penetration depth and the vascular anatomical structure in high-risk facial zones. The static periosteum injection with effective aspiration is recommended as a relatively safe technique. Clinicians are supposed to grasp the anatomy and injection technique to achieve maximum safety during filler injections.

Combination of PDGF-BB and adipose-derived stem cells accelerated wound healing through modulating PTEN/AKT pathway.

Adipose-derived stem cells (ADSCs) have been widely proven to facilitate wound healing. Our study aimed to estimate the influence of combined ADSCs and platelet-derived growth factor-BB (PDGF-BB) on wound healing. We utilized 4 healthy SD rats to isolate ADSCs. Platelet-rich plasma (PRP) was acquired utilizing a two-step centrifugation technology. The role of PRP, PDGF-BB, and PDGF-BB combined with a PI3k inhibitor LY294002 on the viability, migration, and PTEN/AKT pathway in ADSCs were examined utilizing CCK-8, Transwell, and western blot assays. Then, we constructed an open trauma model in SD rats. Effects of ADSCs treated with PDGF-BB on pathological changes, CD31, and PTEN/AKT pathway of wound closure were assessed by hematoxylin & eosin (H&E) staining, Masson staining, immunohistochemical, and western blot assays, respectively. PRP and PDGF-BB intensified the viability and migration of ADSCs by modulating the PTEN/AKT pathway. Interestingly, LY294002 reversed the role of PDGF-BB on ADSCs. In vivo experiments, combined intervention with ADSCs plus PDGF-BB/PRP facilitated wound closure and ameliorated histological injury. Moreover, combined intervention with ADSCs and PDGF-BB attenuated the PTEN level and elevated the CD31 level as well as the ratio of p-AKT/AKT in the skin tissues. A combination of ADSCs and PDGF-BB facilitated wound healing might associate with the regulation of the PTEN/AKT pathway.

The Anatomy Study of Temporal Region based on Ultrasound Investigation: A Spatial Structure Study.

BACKGROUND: With the growing popularity of rejuvenation, people are giving more concerns on their temporal depression which makes them look older and wishing to improve it by injection. The complex structure of the temporal region leads to a higher risk of failed injection. The temporal region is well understood based on cadaver anatomy, but few studies have described its spatial structure. The purpose of this study was to improve the efficacy and safety of temporal injection by studying the spatial structure of the soft tissues and major blood vessels in each layer of the temporal region. METHODS: A total of 30 volunteers (24 men and 6 women, 60 temporal regions) were investigated. Color Doppler ultrasound was used to measure the thickness of the temporal layers at the selected measurement points (A, B, C, D, E, and F). The maximum thickness of the temporal fat pads was also measured, and the layers, depths and diameters of the major temporal vessels (frontal branch of superficial temporal artery and vein, middle temporal vein and deep temporal artery) were measured. RESULTS: At the various measurement points, the thickness and position of the skin, subcutaneous fat superficial fascia, and temporalis muscle did not differ significantly, whereas the superficial temporal fat pad and deep temporal fat pad differed significantly. The diameter and depth of the superficial temporal artery, superficial temporal vein, and deep temporal artery did not differ significantly, whereas the diameter of the middle temporal vein differed slightly, whereas the depth differed more obviously. CONCLUSIONS: The temporal structure is very complex, and understanding the spatial position of each layer of tissue plays an important role in improving the efficacy and safety of temporal filler injection. Ultrasound can help us to understand this information and assist in therapy. LEVEL OF EVIDENCE: Level II.

Effects of inflammation on voriconazole levels: A systematic review.

AIMS: This study aimed to review the studies evaluating the effect of the inflammatory state on voriconazole (VRZ) levels. METHODS: The study included randomized clinical trials, cohort studies, and case-control studies that focused on the influence of the inflammatory state on VRZ levels. Following the preferred reporting items for systematic reviews and meta-analyses (PRISMA) guidelines, relevant articles published until 2021 were searched in several databases, including PubMed, Embase, Web of Science and the Cochrane Library. RESULTS: Twenty studies were included in this review, of which 15 described adult populations, three described paediatric populations, and two included both adult and paediatric populations. Seventeen studies used C-reactive protein (CRP) as an indicator of inflammation, six described a dose-response relationship for the effect of inflammation represented by CRP on VRZ concentrations, and four examined the effect of CRP on the metabolic rate of VRZ. CONCLUSIONS: Our findings showed that the level of inflammation can significantly affect VRZ levels. However, the effect of inflammation on VRZ concentrations in children is controversial and must be analysed along with age. Clinicians dosing VRZ should take into account the patient's inflammatory state. The impact of inflammation on genotype-based dosing decisions requires further study to explain the high pharmacokinetic variability of VRZ.

Novel computer-aided reconstruction of soft tissue defects following resection of oral and oropharyngeal squamous cell carcinoma.

BACKGROUND: Reconstruction of soft tissue defects following surgical tumor resection is important for quality of life in cancer patients with oral and oropharyngeal squamous cell carcinoma (SCC). This study presents a novel computer-aided reconstruction of soft tissue (CARST) technology employed with these patients. METHODS: We first described the CARST technology in detail in a report of a 34-year-old male patient with locally invasive right-sided tongue SCC following a nearly total glossectomy and reported the postoperative outcomes. This digital technology was applied to construct a 3D model from CT images, which was used to delineate surgical resection boundaries and design a personalized reconstruction of the soft tissue defect. A nonuniform rational B-spline (NURBS) was generated and applied to transform the 3D model into a 2D flap-cutting guide printed out using a 3D printer. We then reported a case-series study on oral and oropharyngeal SCC patients who were randomly assigned to receive the CARST (n = 15) or a traditional soft tissue reconstruction (n = 15). Clinicopathological features and short- and long-term postoperative outcomes between the two groups were compared. RESULTS: The patient with the tongue SCC had a successful CARST following surgical tumor resection without any complications. His speech and swallowing functions recovered well after surgery and he experienced no significant changes to his appearance following recovery. There was no recurrence within a 3-year follow-up period. Results of the case-series study showed that the CARST group had significantly shorter operative and post-operation hospital-stay time, a higher flap utilization rate, and a trend of less and milder postoperative complications, and they experienced no significant difference in intraoperative blood loss and long-term outcomes compared to the traditional group. CONCLUSION: CARST is a safer and more efficient personalized technology of soft tissue reconstruction following surgical tumor resection in patients with oral and oropharyngeal SCC.

A New Small-Molecule Compound, Q308, Silences Latent HIV-1 Provirus by Suppressing Tat- and FACT-Mediated Transcription.

Eliminating the latent HIV reservoir remains a difficult problem for creating an HIV functional cure or achieving remission. The "block-and-lock" strategy aims to steadily suppress transcription of the viral reservoir and lock the HIV promoter in deep latency using latency-promoting agents (LPAs). However, to date, most of the investigated LPA candidates are not available for clinical trials, and some of them exhibit immune-related adverse reactions. The discovery and development of new, active, and safe LPA candidates for an HIV cure are necessary to eliminate residual HIV-1 viremia through the block-and-lock strategy. In this study, we demonstrated that a new small-molecule compound, Q308, silenced the HIV-1 provirus by inhibiting Tat-mediated gene transcription and selectively downregulating the expression levels of the facilitated chromatin transcription (FACT) complex. Strikingly, Q308 induced the preferential apoptosis in HIV-1 latently infected cells, indicating that Q308 may reduce the size of the viral reservoir and thus further prevent viral rebound. These findings highlight that Q308 is a novel and safe anti-HIV-1 inhibitor candidate for a functional cure.

Improved viability of murine skin flaps using a gelatin hydrogel sheet impregnated with bFGF.

Basic fibroblast growth factor (bFGF) promotes epithelial cell proliferation and angiogenesis but its clinical applications are limited by its short half-life and low retention. Recently developed gelatin hydrogel sheets able to release physiologically active substances in a controlled manner have the potential to overcome these issues. In this study, the effects of gelatin hydrogel sheets impregnated with bFGF on flap survival and angiogenesis were examined in a murine skin flap model. A flap of 1 × 3 cm was generated on the backs of 60 C57BL/6 mice. The mice were divided into five groups (n = 12/group): Group I, untreated; Group II, treated with a gelatin hydrogel sheet impregnated with saline; Group III, treated with bFGF (50 µg) without sheets; Groups IV and V, treated with gelatin hydrogel sheets impregnated with 50 and 100 µg of bFGF, respectively. On the seventh day after surgery, the flap survival area and vascular network were examined and hematoxylin and eosin and von Willebrand factor staining were used for histological examinations. The flap survival areas were significantly larger in Groups IV and V than in other groups. The area of new vessels was significantly larger in Group IV than in the other groups. In the murine skin flap model, gelatin hydrogel sheets impregnated with bFGF promoted angiogenesis and improved flap survival. These findings support the use of bFGF-impregnated gelatin hydrogel sheets for improving ischemic flap survival in clinical settings.

Preparing Activated Platelet-Rich Plasma for Culturing Human Adipose-Derived Stem Cells.

Activated platelet-rich plasma (PRP) prepared from whole blood via centrifugation demonstrated a proliferation-stimulating effect in several kinds of cultured cells, implying a possible use in regenerative medicine. Here, a double-spin method was used to prepare PRP from whole blood. PRP was further activated by autologous thrombin. The platelet count was measured in the activated PRP and the proliferation-stimulating effect in human adipose-derived stem cells (hASCs) was examined. The resulting platelet count was 11.5-times higher in PRP than in whole blood plasma. The proliferation of hASCs was markedly enhanced by incubation with 1% PRP. The described method can be used to reproducibly prepare PRP with a high concentration of platelets. PRP prepared by this method markedly promotes proliferation of hASCs.

Fibroblast growth factor-2 stimulates proliferation of human adipose-derived stem cells via Src activation.

BACKGROUND: Human adipose-derived stem cells (hASCs) are a subset of mesenchymal stem cells (MSCs); it has been regarded as one of the most promising stem cells. We previously found that fibroblast growth factor-2 (FGF-2) enhanced the proliferation and differentiation of hASC. However, the mechanisms involved in the growth of hASCs by FGF-2 have not been investigated. METHODS: Human adipose-derived stem cells (hASCs) were cultured with FGF-2, and cell growth was assessed. Effects of FGF Receptor (FGFR) inhibitor (NVP-BGJ398), ERK1/2 inhibitor (PD98059), PI3K/Akt inhibitor (LY294002), JNK inhibitor (SP600125), and p38 MAPK inhibitor (SB203580) and Src inhibitor (PP1) on the proliferation were investigated. At the same time, we assessed the effect of FGFR inhibitor on several signaling enzymes such as ERK1/2, JNK, p38, and Akt, in protein level. The involvement of Src activation by FGF-2 was also examined. RESULTS: FGF-2 markedly promoted proliferation of hASCs at concentrations lower than 10 ng/ml and stimulated cell progression to the S and G2/M phases. Proliferation was blocked by the FGFR inhibitor (NVP-BGJ398) and various signaling pathway inhibitors, such as Erk1/2 inhibitor (PD98059), PI3K/Akt inhibitor (LY294002), JNK inhibitor (SP600125), and p38MAPK inhibitor (SB203580). The FGFR inhibitor reduced the activation of protein kinases, such as AKT, Erk1/2, JNK, and p38, in several signaling pathways. The downstream kinase of FGFR, Src, was activated by FGF-2, and its activation was canceled by the FGFR inhibitor. MEK1/2, a downstream kinase of Src, was parallelly regulated by FGF-2. The Src inhibitor (PP1) markedly blocked the proliferation of hASCs via inhibition of Src and MEK1/2. CONCLUSION: Src activation is indispensable for FGF-2-mediated proliferation of ASCs, as well as the subsequent activation of multi-signaling pathways.

A novel bromodomain inhibitor, CPI-203, serves as an HIV-1 latency-reversing agent by activating positive transcription elongation factor b.

The persistence of latent human immunodeficiency virus type 1 (HIV-1) reservoirs remains a major hurdle for HIV-1 eradication. The "shock and kill" strategy relies on the drug-mediated reversion of HIV-1 latency and the subsequent death of HIV-producing cells. Unfortunately, none of the agents currently in use possess a sufficient potency to reactivate latent virus or eliminate the latent HIV-1 reservoir in vivo. Here, we demonstrated that a promising specific bromodomain and extraterminal domain inhibitor, CPI-203, could potently reactivate latent HIV-1 in different latently infected cell lines with minimal cytotoxicity by activating the positive transcription elongation factor b signaling pathway. Notably, CPI-203 exhibited synergism in latent HIV-1 reactivation and alleviated the HIV-1-induced "cytokine storm" when used in combination with the protein kinase C (PKC) agonist prostratin. These findings highlight that CPI-203 shows promise as a novel, safe candidate for the design of targeted strategies to "shock and kill" HIV-1 and thus represents a potential functional cure.

Myricetin antagonizes semen-derived enhancer of viral infection (SEVI) formation and influences its infection-enhancing activity.

BACKGROUND: Semen is a critical vector for human immunodeficiency virus (HIV) sexual transmission and harbors seminal amyloid fibrils that can markedly enhance HIV infection. Semen-derived enhancer of viral infection (SEVI) is one of the best-characterized seminal amyloid fibrils. Due to their highly cationic properties, SEVI fibrils can capture HIV virions, increase viral attachment to target cells, and augment viral fusion. Some studies have reported that myricetin antagonizes amyloid ß-protein (Aß) formation; myricetin also displays strong anti-HIV activity in vitro. RESULTS: Here, we report that myricetin inhibits the formation of SEVI fibrils by binding to the amyloidogenic region of the SEVI precursor peptide (PAP248-286) and disrupting PAP248-286 oligomerization. In addition, myricetin was found to remodel preformed SEVI fibrils and to influence the activity of SEVI in promoting HIV-1 infection. Moreover, myricetin showed synergistic effects against HIV-1 infection in combination with other antiretroviral drugs in semen. CONCLUSIONS: Incorporation of myricetin into a combination bifunctional microbicide with both anti-SEVI and anti-HIV activities is a highly promising approach to preventing sexual transmission of HIV.

Trilobatin as an HIV-1 entry inhibitor targeting the HIV-1 Gp41 envelope.

HIV-1 transmembrane protein gp41 plays a crucial role by forming a stable six-helix bundle during HIV entry. Due to highly conserved sequence of gp41, the development of an effective and safe small-molecule compound targeting gp41 is a good choice. Currently, natural polyanionic ingredients with anti-HIV activities have aroused concern. Here, we first discovered that a glycosylated dihydrochalcone, trilobatin, exhibited broad anti-HIV-1 activity and low cytotoxicity in vitro. Site-directed mutagenesis analysis suggested that the hydrophobic residue (I564) located in gp41 pocket-forming site is pivotal for anti-HIV activity of trilobatin. Furthermore, trilobatin displayed synergistic anti-HIV activities combined with other antiretroviral agents. Trilobatin has a good potential to be developed as a small-molecule HIV-1 entry inhibitor for clinical combination therapy.

Platelet-rich plasma enhances the proliferation of human adipose stem cells through multiple signaling pathways.

BACKGROUND: Platelet-rich plasma (PRP) is an autologous blood product that contains a high concentration of several growth factors. Platelet-derived growth factor (PDGF)-BB is a potential mitogen for human adipose-derived stem cells (hASCs). PRP stimulates proliferation of hASCs; however, the signaling pathways activated by PRP remain unclear. METHODS: hASCs were cultured with or without PRP or PDGF-BB, and proliferation was assessed. hASCs were also treated with PRP or PDGF-BB with or without imatinib, which is a PDGF receptor tyrosine kinase inhibitor, or sorafenib, which is a multikinase inhibitor. Inhibition of cell proliferation was examined using anti-PDGF antibody (Abcam, Cambridge, UK), by cell counting. We assessed the effects of inhibitors of various protein kinases such as ERK1/2, JNK, p38, and Akt on the proliferation of hASCs. RESULTS: The proliferation was remarkably promoted in cells treated with either 1% PRP or 10 ng/ml PDGF-BB, and both imatinib and sorafenib inhibited this proliferation. Anti-PDGF antibody (0.5 and 2 µg/ml) significantly decreased the proliferation of hASCs compared with control. PRP-mediated hASC proliferation was blocked by inhibitors of ERK1/2, Akt, and JNK, but not by an inhibitor of p38. CONCLUSIONS: PRP promotes hASC proliferation, and PDGF-BB in PRP plays a major role in inducing the proliferation of hASCs. PRP promotes hASC proliferation via ERK1/2, PI3K/Akt, and JNK signaling pathways.

Adipose-derived stem cell (ASC)-enriched fat grafting: experiments using White rabbits and an automated cell processing apparatus.

The grafting of fat mixed with adipose-derived stem cells (ASCs) is being increasingly applied to compensate for the disadvantages of previous fat grafting methods. Devices that automatically isolate fat stem cells also have recently been developed. ASCs were isolated from the inguinal region of White rabbits using Icellator®, and the number of cells and their viability were measured. The cell count per fat graft (mL) was adjusted to the following concentrations and subcutaneously transplanted into the back: Control group, Fat + PBS; Fat + ASCs (×0.5) group, 1.6 × 105 cells/mL; and Fat + ASCs (×1) group, 3.2 × 105 cells/mL. Grafted fat weight was measured after 8 weeks, and histological, immunohistological, and specifically stained sections were prepared. Fat absorption was reduced in Fat + ASCs (×0.5) and Fat + ASCs (×1) groups. The number of blood vessels was higher in Fat + ASCs (×1) than in the control group, and blood vessel areas were higher in Fat + ASCs (×0.5) and Fat + ASCs (×1) groups than in the control group. The usefulness of the automated cell processing apparatus, Icellator®, was confirmed, and the results obtained suggest that grafted ASCs promote the vascularization and engraftment of fat grafts.

Platelet-rich plasma stimulates human dermal fibroblast proliferation via a Ras-dependent extracellular signal-regulated kinase 1/2 pathway.

Platelet-rich plasma (PRP) contains a high concentration of several growth factors and contributes to soft-tissue engineering and wound healing. However, the effect of PRP on human dermal fibroblast proliferation and responses is unknown. This was investigated in the present study using PRP prepared from the whole human blood using the double-spin method. Human dermal fibroblast cultures were established from skin samples collected during plastic surgery. Platelet concentration and growth factor levels in PRP were estimated, and a cell proliferation assay was carried out after PRP treatment. The role of Ras-dependent extracellular signal-regulated kinase (ERK)1/2 in the effects of PRP was investigated in human dermal fibroblasts by suppressing ERK1/2 expression with an inhibitor or by short interfering (si)RNA-mediated knockdown, and assessing ERK1/2 phosphorylation by western blotting as well as proliferation in PRP-treated cells. We found that PRP stimulated human dermal fibroblast proliferation, which was suppressed by ERK1/2 inhibitor treatment (P < 0.01). ERK1/2 phosphorylation was increased in the presence of PRP, while siRNA-mediated knockdown of ERK1/2 blocked cell proliferation normally induced by PRP treatment (P < 0.01). These results demonstrate that PRP induces human dermal fibroblast proliferation via activation of ERK1/2 signaling. Our findings provide a basis for the development of agents that can promote wound healing and can be applied to soft-tissue engineering.