Effects of incorporating natural substances into sodium alginate in the cultivation of human dental pulp stem cells: a systematic review with meta-analysis

This study investigated the use of the autologous technology of plasma rich in growth factors (PRGF) as a human-based substitute to fetal bovine serum (FBS) in the culture of human dental pulp stem cells. Stem cell characterization was performed. Analysis of isolation, proliferation, migration, trilineage differentiation, senescence and cryopreservation were compared between FBS and PRGF. Human dental pulp stem cell cultures isolated and maintained with PRGF showed a significantly higher number of cells per explant than FBS cultures. Cell proliferation, migration, osteogenic mineralization and adipogenic differentiation were found to be significantly higher in PRGF than FBS. The autologous PRGF technology could be a suitable and safer substitute for FBS as a culture medium supplement for clinical translation of cell therapy.

P-1017: Using mouse blastocyst as a reprogramming vector for human dental pulp stem cells

PurposeTo investigate whether plasma rich in growth factors (PRGF) eye drops maintain their biological potential after a freeze drying process. The addition of a lyoprotectant like trehalose was also evaluated.MethodsBlood from three healthy donors was collected to obtain eye drops by PRGF technology. The resultant eye drops were divided in four groups: PRGF, freeze-dried PRGF (PRGF lyo), and PRGF lyophilized mixed with 2,5% trehalose (PRGF lyo+2.5T) or 5% trehalose (PRGF lyof+5T). Chemical and biological characteristics were evaluated. Photorefractive keratectomy was performed on C57BL/6 mice which were divided in three treatment groups: control, PRGF, and PRGF lyo. Corneal wound healing and haze formation were evaluated macroscopically. Eyes were collected at 1, 2, 3, and 7 days after surgery, and were processed for histologic studies.ResultsThe pH values of PRGF samples increased significantly after the lyophilization process. Osmolarity levels increased significantly in PRGF samples mixed with trehalose in comparison with PRGF samples without protectants. The freeze drying process maintained growth factors levels as well as the biological properties of PRGF eye drops even without the use of lyoprotectants. PRGF lyo treatment significantly decreased the re-epithelialization time and haze formation in photorefractive keratectomy-treated corneas regarding PRGF and control groups. Furthermore, the PRGF lyo group significantly decreased the number of smooth muscle actin-positive cells in comparison with the control group at each time of the study and at days 2 and 3 in the PRGF group.ConclusionsThe freeze drying process preserves the protein and growth factor content as well as the biological properties of PRGF eye drops, even without the use of protectants. Freeze-dried PRGF eye drops accelerate corneal tissue regeneration after photorefractive keratectomy in comparison with the control group.Translational RelevanceOur study shows the feasibility to preserve the biological capability of PRGF eye drops as freeze-dried formulation, avoiding the addition of protectants.

Human dental pulp stem cells differentiation to neural cells, osteocytes and adipocytes-An in vitro study

Plasma rich in growth factors (PRGF) is a subtype of platelet-rich plasma that has being employed in the clinic due to its capacity to accelerate tissue regeneration. Autologous PRGF has been used in ophthalmology to repair a range of retinal pathologies with some efficiency. In the present study, we have explored the role of PRGF and its effect on microglial motility, as well as its possible pro-inflammatory effects. Organotypic cultures from adult pig retinas were used to test the effect of the PRGF obtained from human as well as pig blood. Microglial migration, as well as gliosis, proliferation and the survival of retinal ganglion cells (RGCs) were analyzed by immunohistochemistry. The cytokines present in these PRGFs were analyzed by multiplex ELISA. In addition, we set out to determine if blocking some of the inflammatory components of PRGF alter its effect on microglial migration. In organotypic cultures, PRGF induces microglial migration to the outer nuclear layers as a sign of inflammation. This phenomenon could be due to the presence of several cytokines in PRGF that were quantified here, such as the major pro-inflammatory cytokines IL-1β, IL-6 and TNFα. Heterologous PRGF (human) and longer periods of cultured (3 days) induced more microglia migration than autologous porcine PRGF. Moreover, the migratory effect of microglia was partially mitigated by: 1) heat inactivation of the PRGF; 2) the presence of dexamethasone; or 3) anti-cytokine factors. Furthermore, PRGF seems not to affect gliosis, proliferation or RGC survival in organotypic cultures of adult porcine retinas. PRGF can trigger an inflammatory response as witnessed by the activation of microglial migration in the retina. This can be prevented by using autologous PRGF or if this is not possible due to autoimmune diseases, by mitigating its inflammatory effect. In addition, PRGF does not increase either the proliferation rate of microglial cells or the survival of neurons. We cannot discard the possible positive effect of microglial cells on retinal function. Further studies should be performed to warrant the use of PRGF on the nervous system.

“Mirror, mirror on the wall, who’s the fairest of them all?“ This is certainly the most cited expression of the 21 century. We do not want to get old, and in this context a lot of hopeful are set on stem cell therapy for tissue regeneration: regeneration of skin, dermis, cartilage, bone, nervous sistem and so on. Most of all on our smile that it is our business card. Rapid and good regeneration of alveolar bone to support new dental implant is up to now ensured by the use adult mesenchymal stem cells. But are they able to conserve their stemness ability for all the age of the donors? In light of such consideration aim of the present work has been the study of the biological properties such as proliferation and stemness ability of human adult dental pulp stem cells (DPSC) in relation to the age of the donors and to the in vitro aging. Human dental pulps derived from adult subjects aged 16–over 66 years have been isolated and cultured in presence of differentiative medium. Results obtained confirmed a correlation between age and conservation of stemness during in vitro aging.

Efficacy and Safety of Plasma Rich in Growth Factors Intra-Articular Infiltrations in the Treatment of Knee Osteoarthritis

Dental pulp is a soft connective tissue that is surrounded by highly mineralized tissues such as enamel, dentin, cementum and provides homeostasis of the tooth. Pulp tissue is a specialized mineralized tissue with a limited regeneration ability due to the anatomical arrangement and postmitotic structure of odontoblastic cells. Classical endodontic treatment is based on removing the infected pulp tissue and root dentin and filling the disinfected canal cavity with a bioinert material to ensure hermetically sealed. It may quickly become necrotic due to tooth decay or inflammation and may require endodontic treatment. In immature that have not completed root formation and necrotic pulp teeth, an apical barrier is formed by using Ca(OH)2 and MTA (Mineral Trioxide Aggregate). There is no increase in root length and thickness in the tooth treated with these methods. For this reason, these teeth remain devital and weak teeth for life. The most desired in endodontic treatment is the replacement of devital and necrotic pulp with healthy pulp tissue. Regenerative endodontic therapy is an alternative to classical endodontic therapy. Regenerative endodontic therapy is defined as '' bio-based procedures designed to treat damaged tooth structures, including cells of the pulp-dentin complex, as well as dentin and root structures. ''Recent advances in biomaterial science and tissue engineering technology have encouraged the development of regenerative endodontic treatment method. Numerous studies are carried out in the field of regenerative endodontic treatment with stem cells, tissue scaffold and growth factors, which are the basis of tissue engineering. Pulp regeneration techniques that are currently proposed are still under development. In this review, studies on different stem cells, growth factors and tissue scaffolds used as the classical tissue engineering trio used in current regenerative endodontics are reviewed and the stages of the treatment procedure developed for routine clinical applications are summarized. In addition, organoids and organ-o-a-chip treatment approaches developed for regenerative endodontic applications were also presented. The relationship between regeneration and cancer was given. Finally, the stages of the treatment procedure developed for routine clinical applications were included.

ABSTRACTBackground:Platelet-rich plasma (PRP) and Plasma Rich in Growth Factors (PRGF) are promising regenerative therapies aimed at enhancing endometrial thickness and improving in vitro fertilization (IVF) outcomes. This study aims to assess the impact of PRP and PRGF on endometrial development, implantation success, and pregnancy rates in IVF patients. A total of 10 ml of venous blood will be drawn from participants and processed to isolate platelets. PRP will be prepared through centrifugation to concentrate platelets, while PRGF will be generated using a leukocyte-poor method and activated with calcium chloride. Endometrial thickness (EMT) will be measured via transvaginal ultrasound (TVS) on the fifth day of the menstrual cycle. If EMT is below 7 mm, PRP or PRGF will be administered, with additional treatments as needed. The study will compare endometrial response, implantation rates, clinical pregnancy rates, and live birth rates between the PRP and PRGF groups. Additionally, safety and potential side effects will be closely monitored to ensure patient well-being.Objectives:The study will assess the impact of PRP and PRGF on endometrial thickness and compare pregnancy outcomes, including clinical pregnancy rates in patients treated with either PRP or PRGF. The study will also evaluate the safety of these treatments and identify any adverse effects associated with them.Materials and Methods:On the 8th day of the menstrual cycle, transvaginal ultrasound (TVS) will assess endometrial thickness. If it’s less than 7 mm, PRP will be injected into the uterine cavity using a PRP catheter. EMT will be reassessed after 48 hours, with additional PRP if needed. Embryo transfer will occur only when EMT reaches 7 mm, using the GnRH antagonist protocol. If EMT exceeds 7 mm during ovarian stimulation, no PRP will be given. After one week of estrogen administration, 1 ml of PRGF will be injected under ultrasound guidance. Three PRGF insertions will constitute one cycle, repeated based on endometrial thickness.Expected Result:Our study demonstrates that PRGF is more effective in increasing endometrium thickness than PRP in patients with thin endometrium.

BackgroundCulture medium enriched with Knockout serum replacement (KSR) can produce in vitro mouse sperm, but it is inefficient, strain-specific and contains bovine products, which limits its use in the human clinic. The study aimed to optimize the culture medium for testicular tissue by using plasma rich in growth factors (PRGF) as a serum supplement, addressing the limitations of KSR.MethodsImmature testicular tissues from NMRI mice were cultured for 14 days to identify the optimal PRGF concentration using histological analysis and tubular integrity scoring. Subsequently, tissues were cultured for 42 days with the optimal PRGF concentration and compared to a control group with 10% KSR, followed by evaluation through histological, tubular integrity, and immunofluorescence assays.ResultsAfter 14 days, 5% PRGF media significantly preserved tubule integrity better than 10% and 20% PRGF, performing similarly to 10% KSR. However, after 42 days, the integrity scoring revealed significantly a higher percentage of well-preserved tubules in 5% PRGF compared to 10% KSR. Additionally, only PRGF supported spermatogenesis to the production of flagellated sperm. Real-time PCR analysis revealed that transcript levels of Plzf, Tekt1, and Tnp1 were significantly elevated in 5% PRGF compared to 10% KSR. Immunofluorescence and quantitative analysis confirmed enhanced spermatogenesis progression in 5% PRGF media, with significantly increased numbers of PLZF + spermatogonia, SYCP3 + spermatocytes, ACRBP + spermatids, and Ki67 + proliferating cells per tubule compared to 10% KSR. Moreover, 5% PRGF showed a significantly lower mean fluorescence intensity of the pro-apoptotic marker Bax, with no significant difference in the anti-apoptotic marker Bcl-2 compared to KSR.ConclusionsThe findings suggest that 5%PRGF is a viable alternative to KSR in mouse testicular tissue cultures, promoting structural integrity and spermatogenesis up to the production of flagellated sperm. The results highlight PRGF’s potential to improve culture media for in vitro sperm production, suggesting promising avenues for future human research.Graphical

Plasma rich in growth factors (PRGF) is a subtype of platelet-rich plasma (PRP) that stimulates tissue regeneration and may promote neuronal survival. It has been employed in ophthalmology to achieve tissue repair in some retinal pathologies, although how PRGF acts in the retina is still poorly understood. As a part of the central nervous system, the retina has limited capacity for repair capacity following damage, and retinal insult can provoke the death of retinal ganglion cells (RGCs), potentially producing irreversible blindness. RGCs are in close contact with glial cells, such as Müller cells, that help maintain homeostasis in the retina. In this study, the aim was to determine whether PRGF can protect RGCs and whether it increases the number of Müller cells. Therefore, PRGF were tested on primary cell cultures of porcine RGCs and Müller cells, as well as on co-cultures of these two cell types. Moreover, the inflammatory component of PRGF was analyzed and the cytokines in the different PRGFs were quantified. In addition, we set out to determine if blocking the inflammatory components of PRGF alters its effect on the cells in culture. The presence of PRGF compromises RGC survival in pure cultures and in co-culture with Müller cells, but this effect was reversed by heat-inactivation of the PRGF. The detrimental effect of PRGF on RGCs could be in part due to the presence of cytokines and specifically, to the presence of pro-inflammatory cytokines that compromise their survival. However, other factors are likely to be present in the PRGF that have a deleterious effect on the RGCs since the exposure to antibodies against these cytokines were insufficient to protect RGCs. Moreover, PRGF promotes Müller cell survival. In conclusion, PRGF hinders the survival of RGCs in the presence or absence of Müller cells, yet it promotes Müller cell survival that could be the reason of retina healing observed in the in vivo treatments, with some cytokines possibly implicated. Although PRGF could stimulate tissue regeneration, further studies should be performed to evaluate the effect of PRGF on neurons and the implication of its potential inflammatory role in such processes.

Plasma rich in growth factors (PRGF) eye drops stimulates scarless regeneration compared to autologous serum in the ocular surface stromal fibroblasts

Objectives:In regenerative dentistry, cell proliferation is crucial for tissue repair and immune response modulation, essential for successful regeneration. The adipose-derived stromal vascular fraction (AD-SVF) shows promise in tissue engineering as an autologous therapy. AD-SVF exosomes, cell-free and resilient in ischemic conditions, offer an ethical and hopeful strategy for dental tissue regeneration and wider regenerative medicine use. Although the enhancement of human dental pulp stem cells (hDPSCs) migratory abilities by AD-SVF exosomes is known, their impact on hDPSC proliferation requires further examination.Aims:This study explores how AD-SVF exosomes influence hDPSC proliferation.Methods:AD-SVF exosomes, isolated using size exclusion chromatography and characterized through flow cytometry and nanoparticle tracking analysis, were used to treat hDPSCs at varying concentrations. Proliferation was assessed with the cell counting kit-8 assay. Statistical analysis involved one-way ANOVA, post hoc LSD testing, with significance at 0.05.Results and Conclusion:Isolated AD-SVF exosomes, averaging 103 ± 24 nm, expressed CD9+ and CD63+ markers. The study revealed increased hDPSC proliferation on the 5th day post low-exosome treatment, indicating a positive association between AD-SVF exosomes and cell proliferation. These results highlight the potential of AD-SVF exosomes to enhance hDPSC proliferation for dental pulp and broader tissue regeneration.

The use of plasma rich in growth factors (PRGF) has gained importance in many medical fields due to its regenerative potential. The aim of this study is to evaluate the effects of PRGF on primary skin fibroblasts assessing cell proliferation, migration and secretion of growth factors. The age of the patients from who PRGF was prepared was also studied to determine whether it influenced the outcomes. Human dermal fibroblasts were isolated from three healthy volunteers. Using PRGF-Endoret technology, PRGF was prepared from two groups of different ages (18-35 years and 50+ years). The effects of increasing concentration of PRGF (5%, 10% and 20%) on cell proliferation and migration was evaluated. Biosynthetic behaviour of cells was also analysed measuring vascular endothelial growth factor (VEGF), transforming growth factor b1 (TGFb1) and pro-collagen type I secreted levels with or without PRGF treatment. Mean platelet enrichment reached 2.4X and 2X in 18-35 and 50+ groups respectively. A dose-dependent response was observed in proliferation assays achieving the highest levels with 20% PRGF. Migration was also promoted in cells but not in a dose-dependent manner. Cell proliferation and migration outcomes obtained with PRGF (from both groups) were significantly higher compared to non-stimulated groups (p<0.05), with no statistical significances were observed between the different age groups. Production of VEGF, TGFb and procollagen type I was significantly increased by cells treated with PRGF, however, with the exception of VEGF, no statistical significances were observed between the different age groups. Results from this study concluded that PRGF is safe and effective in stimulating skin regeneration by enhancing proliferation, migration and expression of pivotal bioactive molecules involved in wound healing and haemostasis.

Dental pulp represents a promising and easily accessible source of mesenchymal stem cells for clinical applications. Many studies have investigated the use of human dental pulp stem cells and stem cells isolated from the dental pulp of human exfoliated deciduous teeth for bone tissue engineering in vivo. However, the type of scaffold used to support the proliferation and differentiation of dental stem cells, the animal model, the type of bone defect created, and the methods for evaluation of results were extremely heterogeneous among these studies conducted. With this issue in mind, the main objective of this study is to present and summarize, through a systematic review of the literature, in vivo studies in which the efficacy of human dental pulp stem cells and stem cells from human exfoliated deciduous teeth (SHED) for bone regeneration was evaluated. The article search was conducted in PubMed/MEDLINE and Web of Science databases. Original research articles assessing potential of human dental pulp stem cells and SHED for in vivo bone tissue engineering, published from 1984 to November 2017, were selected and evaluated in this review according to the following eligibility criteria: published in English, assessing dental stem cells of human origin and evaluating in vivo bone tissue formation in animal models or in humans. From the initial 1576 potentially relevant articles identified, 128 were excluded due to the fact that they were duplicates and 1392 were considered ineligible as they did not meet the inclusion criteria. As a result, 56 articles remained and were fully analyzed in this systematic review. The results obtained in this systematic review open new avenues to perform bone tissue engineering for patients with bone defects and emphasize the importance of using human dental pulp stem cells and SHED to repair actual bone defects in an appropriate animal model.