Plasma Rich in Growth Factors in Bone Regeneration: The Proximity to the Clot as a Differential Factor in Osteoblast Cell Behaviour.

The osteogenic differentiation process, by which bone marrow mesenchymal stem cells and osteoprogenitors transform into osteoblasts, is regulated by several growth factors, cytokines, and hormones. Plasma Rich in Growth Factors (PRGF) is a blood-derived preparation consisting of a plethora of bioactive molecules, also susceptible to containing epigenetic factors such as ncRNAs and EVs, that stimulates tissue regeneration. The aim of this study was to investigate the effect of the PRGF clot formulation on osteogenic differentiation. Firstly, osteoblast cells were isolated and characterised. The proliferation of bone cells cultured onto PRGF clots or treated with PRGF supernatant was determined. Moreover, the gene expression of Runx2 (ID: 860), SP7 (ID: 121340), and ALPL (ID: 249) was analysed by one-step real-time quantitative polymerase chain reaction (RT-qPCR). Additionally, alkaline phosphatase (ALPL) activity determination was performed. The highest proliferative effect was achieved by the PRGF supernatant in all the study periods analysed. Concerning gene expression, the logRGE of Runx2 increased significantly in osteoblasts cultured with PRGF formulations compared with the control group, while that of SP7 increased significantly in osteoblasts grown on the PRGF clots. On the other hand, despite the fact that the PRGF supernatant induced ALPL up-regulation, significantly higher enzyme activity was detected for the PRGF clots in comparison with the supernatant formulation. According to our results, contact with the PRGF clot could promote a more advanced phase in the osteogenic process, associated to higher levels of ALPL activity. Furthermore, the PRGF clot releasate stimulated a higher proliferation rate in addition to reduced SP7 expression in the cells located at a distant ubication, leading to a less mature osteoblast stage. Thus, the spatial relationship between the PRGF clot and the osteoprogenitors cells could be a factor that influences regenerative outcomes.

Improving the mechanical and biological functions of cell sheet constructs: The interplay of human-derived periodontal ligament stem cells, endothelial cells and plasma rich in growth factors.

OBJECTIVE: The aim of this study was to produce and characterize triple-layered cell sheet constructs with varying cell compositions combined or not with the fibrin membrane scaffold obtained by the technology of Plasma Rich in Growth Factors (mPRGF). MATERIALS AND METHODS: Human primary cultures of periodontal ligament stem cells (hPDLSCs) were isolated, and their stemness nature was evaluated. Three types of triple-layered composite constructs were generated, composed solely of hPDLSCs or combined with human umbilical vein endothelial cells (HUVECs), either as a sandwiched endothelial layer or as coculture sheets of both cell phenotypes. These three triple-layered constructs were also manufactured using mPRGF as cell sheets' support. Necrosis, glucose consumption, secretion of extracellular matrix proteins and synthesis of proangiogenic factors were determined. Histological evaluations and proteomic analyses were also performed. RESULTS: The inclusion of HUVECs did not clearly improve the properties of the multilayered constructs and yet hindered their optimal conformation. The presence of mPRGF prevented the shrinkage of cell sheets, stimulated the metabolic activity and increased the matrix synthesis. At the proteome level, mPRGF conferred a dramatic advantage to the hPDLSC constructs in their ability to provide a suitable environment for tissue regeneration by inducing the expression of proteins necessary for bone morphogenesis and cellular proliferation. CONCLUSIONS: hPDLSCs' triple-layer construct onto mPRGF emerges as the optimal structure for its use in regenerative therapeutics. CLINICAL RELEVANCE: These results suggest the suitability of mPRGF as a promising tool to support cell sheet formation by improving their handling and biological functions.

Biological and Adhesive Properties of an Autologous Protein-Based Fibrin Sealant for Ophthalmological Applications.

Purpose: The aim of this study was to evaluate the biological and adhesive properties of a new autologous sealant based on plasma rich in growth factors (PRGF), named E-Sealant. Methods: Conventional PRGF and a commercial fibrin sealant (Tisseel) were included as controls. The hematological and protein content of E-Sealant was determined. Its bioactivity and biocompatibility were tested for human keratocytes (HKs). To evaluate its adhesion and regenerative capacity, E-Sealant was used on an animal model of conjunctival autograft surgery and compared to Tisseel. Results: E-Sealant presented a high growth factor content with levels similar to those of conventional PRGF. E-Sealant induced proliferative and migratory activity on HK cells equivalent to PRGF. Although autologous membranes induced the proliferation of HKs, cells cultured over Tisseel did not adhere nor proliferate. HK cells showed increased number and flattened morphology over PRGF and E-Sealant compared to scarce and round-shape cells detected in Tisseel. Conjunctival autograft glued with E-Sealant adhered successfully, whereas Tisseel application formed irregular clots. During follow-up, both adhesives showed good integration and no dehiscence. However, Tisseel-treated samples presented slightly increased hemorrhage and inflammation. In contrast to Tisseel, E-Sealant-treated autografts presented a continuous layer of non-keratinized stratified squamous epithelium. Inflammatory infiltrates were minimal in E-Sealant-treated conjunctiva, whereas the Tisseel group showed noticeable immune reactions. Unlike Tisseel-treated grafts, E-Sealant presented low immunoreactivity for smooth muscle actin (SMA), suggesting decreased fibrotic tissue formation. Conclusions: E-Sealant presents optimal biological and adhesive properties suitable for use as an ophthalmic glue, with regenerative purposes superior to commercial fibrin sealants. Translational Relevance: Our study analyzed the characterization and biological activity of a new autologous fibrin sealant in ocular surface cells and in an animal model in which the adhesive and regenerative properties of the fibrin sealant were evaluated.

Cellular composition modifies the biological properties and stability of platelet rich plasma membranes for tissue engineering.

Scaffolds should provide structural support for tissue regeneration, allowing their gradual biodegradation and interacting with cells and bioactive molecules to promote remodeling. Thus, the scaffold's intrinsic properties affect cellular processes involved in tissue regeneration, including migration, proliferation, differentiation, and protein synthesis. In this sense, due to its biological effect and clinical potential, Platelet Rich Plasma (PRP) fibrin could be considered a successful scaffold. Given the high variability in commercial PRPs formulations, this research focused on assessing the influence of cellular composition on fibrin membrane stability and remodeling cell activity. The stability and biological effect were evaluated at different time points via D-dimer, type I collagen and elastase quantification in culture media conditioned by Plasma Rich in Growth Factors - Fraction 1 (PRGF-F1), Plasma Rich in Growth Factors - Whole Plasma (PRGF-WP) and Leukocyte-rich Platelet Rich Plasma (L-PRP) membranes, and by gingival fibroblast cells seeded on them, respectively. Ultrastructure of PRP membranes was also evaluated. Histological analyses were performed after 5 and 18 days. Additionally, the effect of fibrin membranes on cell proliferation was determined. According to the results, L-PRP fibrin membranes degradation was complete at the end of the study, while PRGF membranes remained practically unchanged. Considering fibroblast behavior, PRGF membranes, in contrast to L-PRP ones, promoted extracellular matrix biosynthesis at the same time as fibrinolysis and enhanced cell proliferation. In conclusion, leukocytes in PRP fibrin membranes drastically reduce scaffold stability and induce behavioral changes in fibroblasts by reducing their proliferation rate and remodeling ability.

Current opinion on the role of vitamin D supplementation in respiratory infections and asthma/COPD exacerbations: A need to establish publication guidelines for overcoming the unpublished data.

BACKGROUND & AIMS: The role of prophylactic vitamin D supplementation in prevention and treatment of respiratory infections and other related pathologies has been extensively explored with conflicting results. The aim of this systematic review and meta-analysis was to evaluate the prophylactic and therapeutic effects of vitamin D administration on respiratory infections. METHODS: A systematic search was performed and randomized controlled trials (RCTs) of vitamin D supplementation and a total of 65 RCTs involving 50,554 participants were included. RESULTS: The overall incidence of respiratory infections in terms of count data (OR: 0.87; 95%CI [0.80-0.95]; p = 0.0028; I2 = 43%) and event rate (IRR: 0.81; 95%CI [0.70-0.95]; p = 0.010; I2 = 79%) was significantly reduced in the intervention group. However, no effect of vitamin D on duration or upper respiratory tract infection severity was observed following the overall analysis. Subgroup analyses revealed more details regarding the protective effect of particular dose ranges, administration frequencies and trial durations on different disease types. CONCLUSIONS: Despite between-study heterogeneity was high for most outcomes and publication bias may have led to an effect size overestimation of incidence count data, vitamin D supplementation could be beneficial in improving resistance to overall respiratory infections, particularly when administered on a daily basis.

In vitro and in vivo Effect of Platelet-Rich Plasma-Based Autologous Topical Serum on Cutaneous Wound Healing.

INTRODUCTION: Skin injury and wound healing is an inevitable event during lifetime. However, several complications may hamper the regeneration of the cutaneous tissue and lead to a chronic profile that prolongs patient recovery. Platelet-rich plasma is rising as an effective and safe alternative to the management of wounds. However, this technology presents some limitations such as the need for repeated blood extractions and health-care interventions. OBJECTIVE: The aim of this study was to assess the use of an endogenous and storable topical serum (ES) derived from plasma rich in growth factors promoting wound healing, and to obtain preliminary data regarding its clinical and experimental effect over ulcerated skin models and patient care. METHODS: Human dermal fibroblast and 3D organotypic ulcerated skin models were used to assess ES over the main mechanisms of wound healing including cell migration, edge contraction, collagen synthesis, tissue damage, extracellular matrix remodeling, cell death, metabolic activity, and histomorphometry analysis. Additionally, 4 patients suffering from skin wounds were treated and clinically assessed. RESULTS: ES promoted dermal fibroblast migration, wound edge contraction, and collagen synthesis. When topically applied, ES increased collagen and elastin deposition and reduced tissue damage. The interstitial edema, structural integrity, and cell activity were also maintained, and apoptotic levels were reduced. Patients suffering from hard-to-heal wounds of different etiologies were treated with ES, and the ulcers healed completely within few weeks with no reported adverse events. CONCLUSION: This preliminary study suggests that ES might promote cutaneous wound healing and may be useful for accelerating the re-epithelization of skin ulcers.

The inclusion of leukocytes into platelet rich plasma reduces scaffold stability and hinders extracellular matrix remodelling.

BACKGROUND: Scaffolds should have controllable degradation rate and allow cells to produce their own extracellular matrix. Platelet rich plasma (PRP) is a source of autologous growth factors and proteins embedded in a 3D fibrin scaffold. There is no consensus regarding the obtaining conditions and composition of PRPs. The aim of this study was to evaluate how the inclusion of leukocytes (L-PRP) in plasma rich in growth factors (PRGF) may alter the process of fibrinolysis. The effect of different combinations of cellular phenotypes with PRGF and L-PRP clots on both the fibrinolysis and matrix deposition process was also determined. METHODS: PRGF and L-PRP clots were incubated for 14 days and D-dimer and type I collagen were determined in their conditioned media to evaluate clots' stability. For remodelling assays, gingival fibroblasts, alveolar osteoblasts and human umbilical vein endothelial cells (HUVEC) were seeded onto the two types of clots for 14 days. D-dimer, type I collagen, and laminin α4 were measured by ELISA kits in their conditioned media. Morphological and histological analysis were also performed. Cell proliferation was additionally determined RESULTS: PRGF clots preserved their stability as shown by the low levels of both D-dimer and collagen type I compared to those obtained for L-PRP clots. The inclusion of both gingival fibroblasts and alveolar osteoblasts stimulated a higher fibrinolysis in the PRGF clots. In contrast to this, the degradation rates of both PRGF and L-PRP clots remained unchanged after culturing with the endothelial cells. In all cases, type I collagen and laminin α4 levels were in line with the degree of clots' degradation. In all phenotypes, cell proliferation was significantly higher in PRGF than in L-PRP clots. CONCLUSION: The inclusion of leukocytes in PRGF scaffolds reduced their stability, decreased cell number and slowed down cell remodelling.

The Effectiveness of Platelet-Rich Plasma as a Carrier of Stem Cells in Tissue Regeneration: A Systematic Review of Pre-Clinical Research.

Fibrin, as a physiological scaffold, presents many advantages compared to synthetic materials, such as controllable degradation, non-toxic byproducts, and excellent biocompatibility. The use of stem cells along with the biomimicking scaffolds and signalling factors would complete the triad of the tissue engineering approach. In this context, the aim of this narrative review is to compile the current evidence in vitro and in vivo regarding the combination of platelet-rich plasma (PRP) scaffolds and adult stem cells focusing on the regenerative medicine field according to the clinical application. A total of 1,832 articles were identified during the systematic literature search through three databases (Google Scholar, PubMed, and Web of Science). A total of 18 publications reaching the inclusion criteria were finally included in this review. According to the results, the combination of PRP and stem cells improved osteogenic and cartilage regeneration. However, regarding periodontal regeneration conflicting results were reported. Many other pathologies were also investigated, and even though no general conclusions were able to be obtained, most of them supported the superior performance of PRP-based scaffolds seeded with stem cells. Finally, the lack of a comprehensive description of the PRP composition makes it difficult to compare them and to draw robust conclusions. Therefore, an accurate and complete description of the product needs to be detailed in upcoming scientific publications.

A Novel Autologous Topical Serum Based on Plasma Rich in Growth Factors Technology Counteracts Ultraviolet Light-Derived Photo-Oxidative Stress.

INTRODUCTION: Skin as the major barrier between the internal and external environments protects our body from external injuries. Ultraviolet B (UVB) radiation is majorly responsible for photoaging and is closely associated with oxidative stress, inflammation, and DNA damage. The progression in the field of biological therapies has led to the emergence of new autologous therapies based on growth factors. An autologous topical serum (ATS) based on the plasma rich in growth factors (PRGF) technology has also been developed with regenerative properties. OBJECTIVE: The aim of this study was to evaluate this new topical formulation in protecting skin against UVB-induced photodamage using dermal fibroblast cultures and 3D skin models. METHODS: ATS was assessed over the main mechanisms underlying photodamage including oxidative stress, cell viability, DNA damage, cell death, and biosynthetic activity. Three different irradiation protocols were tested. RESULTS: ATS application showed to significantly reduce free radical production and cell death caused by ultraviolet radiation. It also increased cell viability and promoted the proliferative activity and fibronectin biosynthesis of dermal fibroblasts. DNA double-strand cleavage that occurs after photo-oxidative stress was reduced. Photoexposed 3D explants showed higher levels of metabolic activity and collagen synthesis. Histomorphometric analysis also revealed a reduction in UV-derived edema, hyperkeratosis, and apoptosis and an increase in collagen and cell bioactivity. CONCLUSION: This preliminary study suggests that this novel ATS might counteract the harmful effects of UV radiation.

The influence of alveolar bone healing degree on its potential as a source of human alveolar bone-derived cells.

BACKGROUND: The concept of bone tissue engineering has emerged as a novel alternative approach that comprises three essential components: osteogenic cells, osteoinductive signals and osteoconductive scaffolds. The low-speed drilling represents a useful and accessible autologous source for human alveolar bone-derived cells (hABCs). The aim of this study was to compare the efficacy of two donor sites (healing sites (HS) and non-augmented healed sites (NAHS)) as a source of hABCs. METHODS: Nineteen patients were enrolled in this study. The patients' demographic data were described. Bone type and dental implant location were also determined. The hABCs obtained were characterized. Apoptosis and sclerostin expression in the samples were also assessed with immunohistochemistry. RESULTS: The hABCs left earlier the tissue explants of the HS than the NAHS. The proliferation of the hABCs had reached the sub-confluence stage in both groups. Cellular efficacy was not statistically significant between the two groups. The hABCs exhibited osteogenic phenotype as they expressed bone sialoprotein (BSP), osteopontin (OP) and tissue non-specific alkaline phosphatase (TNAP). In both groups, the level and the distribution pattern of apoptotic cells and sclerostin expression were similar. CONCLUSIONS: Within the limitations of this study, both HS and NAHS were similarly effective to provide hABCs.

Phytochemicals determination and classification in purple and red fleshed potato tubers by analytical methods and near infrared spectroscopy.

BACKGROUND: Over the last two decades, the attractive colours and shapes of pigmented tubers and the increasing concern about the relationship between nutrition and health have contributed to the expansion of their consumption and a specialty market. Thus, we have quantified the concentration of health promoting compounds such as soluble phenolics, monomeric anthocyanins, carotenoids, vitamin C, and hydrophilic antioxidant capacity, in a collection of 18 purple- and red-fleshed potato accessions. RESULTS: Cultivars and breeding lines high in vitamin C, such as Blue Congo, Morada and Kasta, have been identified. Deep purple cultivars Violet Queen, Purple Peruvian and Vitelotte showed high levels of soluble phenolics, monomeric anthocyanins, and hydrophilic antioxidant capacity, whereas relatively high carotenoid concentrations were found in partially yellow coloured tubers, such as Morada, Highland Burgundy Red, and Violet Queen. CONCLUSION: The present characterisation of cultivars and breeding lines with high concentrations of phytochemicals is an important step both to support the consideration of specialty potatoes as a source of healthy compounds, and to obtain new cultivars with positive nutritional characteristics. Moreover, by using near infrared spectroscopy a non-destructive identification and classification of samples with different levels of phytochemicals is achieved, offering an unquestionable contribution to the potato industry for future automatic discrimination of varieties.