Bone regeneration in osteoporosis by delivery BMP-2 and PRGF from tetronic–alginate composite thermogel

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.

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.

Plasma rich in growth factors (PRGF) had been used in regenerative therapy such as soft tissue and newly bone formation. However, there has been little basic research into the efficacy of PRGF in bone regeneration. In this study, we evaluated the efficacy for new bone formation by transplantation of PRGF onto rat calvaria. The osteogenic potential was evaluated by histologic findings, immunohistochemistry and bone formation analyzed with micro-computed tomography (micro-CT). PRGF was prepared by centrifugation of rat whole blood (WB), and then was activated using 10% calcium chloride solution. The activated PRGF transplanted within a polytetrafluoroethylene (PTFE) tube was transplanted onto calvarial bone of rats. Histological observation demonstrated that PRGF group showed newly formed bone in a wide range. Immunohistochemistry showed Runx2, Osterix, Bone Alkaline Phosphatase (BAP) and Osteocalcin expressed in PRGF group during the early stage of bone formation. Micro-CT showed that PRGF group promoted an increase in bone volume which compared to control group. We concluded that PRGF has more capacity for bone regeneration, and PRGF may be useful in bone regeneration treatment.

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.

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

Vascular endothelial growth factor (VEGF)-A, the most abundant subtype of the VEGF family in the eye, plays an important role in corneal homeostasis due to its ability to mediate corneal nerve repair. Repeated intravitreal anti-VEGF injections were shown to significantly reduce corneal nerve density, which might negatively affect corneal homeostasis and lead to a neuropathic dry eye disease. Currently, there are two effective modalities to treat dry eye while supplying VEGF to the ocular surface: serum eye drops (SED) and eye drops manufactured from plasma rich in growth factors (PRGF). The purpose of this study was to measure the VEGF-A concentration in SED and PRGF eye drops. Ten healthy volunteers donated blood on two separate occasions, 2 - 8 days apart. Thus, a total of 20 blood samples were processed to obtain both SED and PRGF. Concentrations of VEGF-A were quantified by a Simple Plex platform run in triplicate. The VEGF-A concentration in SED and PRGF was very similar between the two blood samples drawn from one individual donor but showed substantial interindividual variability. However, in all 20 samples, VEGF concentrations were substantially higher in SED samples (mean 238.7 ± 146.6 pg/mL) compared to PRGF samples (mean 67.4 ± 46.3 pg/mL). Based upon the analysis of variance (ANOVA) model for the measured concentrations with fixed effects for specimen (SED vs. PRGF) and subject, the mean difference between the SED and PRGF concentration was 168.1 pg/mL (95% confidence interval: [142.4, 193.9], p < 0.001). Our study showed that the VEGF concentration was higher in SED than in PRGF. This is an important finding, particularly for potential treatment of dry eye disease in patients with neuropathic eye disease, especially in patients that received repeated anti-VEGF intravitreal injections, or in patients with Sjögren's disease, where the level of VEGF in tears might be pathologically decreased. Hypothetically, VEGF might be needed to restore ocular surface homeostasis. Although growing evidence has shown that VEGF-A plays an important role in corneal homeostasis, only a randomized prospective clinical trial will show whether supplying VEGF-A to the ocular surface might successfully restore the corneal homeostasis and overcome the problem of corneal neuropathy in these patients. For such a trial, based on our results, an undiluted SED should be preferred over a PRGF due to the higher content of VEGF-A.

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.

Aim: The aim of this study was to evaluate the osteogenic potential of adipose-derived stem cells (ADSCs) and to assess the influence of plasma rich in growth factors (PRGF) on bone regeneration using ADSCs. Materials & methods: Bone defects were randomly allocated to the five treatment modalities: spontaneous healing, natural bovine bone mineral (BBM), BBM loaded with PRGF, BBM loaded with ADSCs and BBM loaded with a combination of ADSCs and PRGF. Results: The PRGF significantly enhanced the biomaterial-to-bone contact. Defects treated with ADSCs and PRGF or a combination of both showed the greatest bone regeneration. Conclusion: Combining PRGF and ADSCs boosts the bone graft regenerative potential at the earliest period of healing.

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

BackgroundThe use of autologous platelet-rich plasma (PRP) and plasma rich in growth factors (PRGF) has been proposed for the treatment of several acute and chronic syndromes, such as corneal epithelial defects and dry eye syndrome, gum bleeding during oral surgery, and in orthopaedic surgery. We hypothesized that PRGF, rather than PRP, could be more effective because of its intrinsic characteristics in promoting the healing of intestinal anastomosis. The purpose of the present study was to evaluate and compare the effects of PRP and PRGF on various parameters of anastomotic healing in a swine model.MethodsEight female pigs were randomly assigned to two groups and subjected to hand sewn jeujuno-jejunal appositional extramucosal anastomoses. For each animal, a total of six anastomoses were performed: two were considered controls and received no treatment, while the remaining four anastomoses were treated with PRP or PRGF of which both were prepared at a platelet concentration that was respectively 3.4-fold and 2.81-fold higher than the original platelet count. In each animal, either PRP or PRGF was used as a treatment, to avoid interference among products. Animals were euthanized after 8 days and the anastomoses were evaluated and compared for the presence of adhesions, anastomotic leakage, bursting pressure, and histological appearance.ResultsThe concentration of platelets in PRP was 3.41-fold higher (range, 3.20–4.24) that the concentration in whole blood, while the concentration in PRGF was 2.81-fold higher (range, 2.89–4.88).The results obtained from the present study highlighted that there are no differences between anastomotic samples treated with either PRP or PRGF preparations, except for a significant increase in epithelization of the intestinal mucosa at the anastomotic site in the PRGF group.ConclusionsBoth PRP and PRGF suspensions should be considered a safe strategy and represent a relatively low-cost technology that is flexible enough to be applied in several therapeutic fields. No true benefit could be proven in our study compared to the no treatment following anastomoses formation, with the exception of enhanced epithelization of the mucosa in the PRGF group.

Purpose: To evaluate the utility and safety of plasma rich in growth factors (PRGF) eye drops in the postoperative treatment of non‐penetrating deep sclerectomy (NPDS).Methods: Retrospective case–control study, including open‐angle glaucoma patients. Cases were consecutively recruited, and used topical PRGF for 4 months after NPDS. Historical controls were patients who had undergone the same technique immediately before the start of the study (without PRGF). Prior to surgery, intraocular pressure (IOP), the number of topical hypotensive medications, and visual field mean deviation (MD) were registered. Postoperatively (1 week, 3 and 6 months), those three variables were also analysed, as well as complications, manipulations and reinterventions.Results: Preoperatively, the PRGF group (n = 37) and the control group (n = 49) were similar in age (70.9 ± 10.0 vs. 71.5 ± 10.7 years; p = 0.68), IOP (23.0 ± 9.0 vs. 20.6 ± 10.2 mmHg; p = 0.26), MD (−15.0 ± 8.8 vs. −15.9 ± 7.8 dB; p = 0.76) and the number of hypotensive medications (2.8 ± 0.9 vs. 2.7 ± 0.8; p = 0.40). IOP significantly decreased at all follow‐up visits in both groups, compared to preoperative values. That reduction was greater at 6 months with PRGF (p &lt; 0.01): 10.9 ± 4.3 mmHg (−52.6%) versus 15.0 ± 8.0 mmHg (−27.2%). In the final analysis, the number of drugs similarly reduced in both groups (p = 0.32). MD showed no significant differences. Surgical complications were observed in 12 control eyes (25%) and 5 eyes (14%) treated with PRGF (p = 0.06). No complications related to the use of PRGF were identified. The needling rates were 9.4% and 5.9% in PRGF and controls, respectively (p = 0.58). Three eyes (6.3%) required further glaucoma surgery in the control group, and 1 (2.7%) in the PRGF‐group (p = 0.78). The final complete success rate was higher with PRGF: 83.8% vs 60.4%.Conclusions: Postoperative PRGF reduced IOP and complications rate in the mid‐term after NPDS, thus it may be considered as a possible safe adjuvant agent to achieve surgical success.

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.

Objectives: To assess if the instillation of plasma rich growth factors (PRGFs) improves endometrial thickness (EMT) in frozen embryo cycles performed under hormone treatment where the endometrium was very thin (≤5 mm). Methods: First, a randomized controlled trial (RCT) was performed comparing women only receiving an increase in estrogen therapy (n = 9) and women receiving both the increase in estrogen therapy and three instillations of PRGF (n = 13). The second part of the study consisted of a prospective observational follow-up of the patients included in the RCT (for 1-3 months in the study group, and for 1-6 months in the control group). Results: In the RCT, there was an increase in EMT in both the PRGF and control groups. However, the increase was significantly higher in the PRGF group (1.30 ± 0.67 mm) compared to the control group (0.58 ± 0.51 mm). In the PRGF group, 23% achieved an EMT of 7 mm compared to 0% in the control group. There were 2 pregnancies in the PRGF group resulting from the 3 transfers performed in that group. No transfer was carried out in the control group. There was a significant increase in EMT 1-3 months after the PRGF cycle. The live birth rate per transfer was 20% in the cycles following the PRGF cycles, whereas it was 30% per starting woman. For women in the control group who later underwent PRGF, the LBR was 57.1%. Conclusions: PRGF instillation in cases of very thin endometrium increases EMT moderately. It is suggested that the beneficial effect of PRGF may persist for 1-3 cycles after instillation.

Background and Objectives: The aim of this study is to employ quantitative proteomics to elucidate the molecular mechanism and signaling pathways modulated by plasma rich in growth factors (PRGF) in a murine model of geographic atrophy (GA)-like retinal degeneration. Materials and Methods: C57BL/6J mice were used as a model GA-like retinal degeneration by a single systemic NaIO3 administration. Animals were divided into three groups: Control (PBS), Disease (NaIO3 + PBS), and PRGF-treated (NaIO3 + PRGF). After 7 days, retinas and retinal pigment epithelium were collected for proteomic analysis. Proteins were extracted, digested using the FASP method, and analyzed by Data-Independent Acquisition (DIA-PASEF) mass spectrometry; data were processed with DIA-NN and statistically analyzed with Perseus. Functional pathway analysis was performed using Ingenuity Pathway Analysis. Results: A total of 6511 proteins were identified. The Disease model showed the expected deregulation of pathways related to oxidative stress, inflammation, and fibrosis. Comparison between the PRGF and Control groups showed that PRGF significantly reduced oxidative and cellular stress proteins/pathways. In the same way, when PRGF and Disease groups were compared, PRGF treatment showed a significant reduction in pathways associated with inflammation, oxidative stress, and cellular stress. PRGF also activated several homeostatic pathways not only related to neuroprotective pathways but also with the lipid deposition (drusen) reduction. All these results suggest that PRGF treatment exerts a protective effect against NaIO3-induced retinal damage. Conclusions: These findings suggest that PRGF effectively mitigates the degenerative effects of NaIO3 by activating specific protective and compensatory signaling pathways in the retina. PRGF is indicated as a promising new therapeutic option for ameliorating age-related macular degeneration progression.

Objective:Reconstruction methods are an essential prerequisite for functional rehabilitation of the stomatognathic system. Plasma rich in growth factors (PRGF) offers a new and potentially useful adjunct to bone substitute materials in bone reconstructive surgery. This study was carried out to investigate the influence of PRGF and fibrin membrane on regeneration of bony defects with and without deproteinized bovine bone mineral (DBBM) on rabbit calvaria.Materials and Methods:Twelve New Zealand white rabbits were included in this randomized, blinded, prospective study. Four equal 3.3×6.6 mm cranial bone defects were created and immediately grafted with DBBM, PRGF+DBBM, PRGF+fibrin membrane and no treatment as control. The defects were evaluated with histologic and histomorphometric analysis performed 4 and 8 weeks later.Results:Adding PRGF to DBBM led to increased bone formation as compared with the control group in 4- and 8-week intervals. In DBBM and PRGF+fibrin membrane samples, no significant increase was seen compared to the control group. There was also a significant increase in the rate of biodegradation of DBBM particles with the addition of PRGF in the 8-week interval. Neither noticeable foreign body reaction nor any severe inflammation was seen in each of the specimens evaluated.Conclusion:Under the limitation of this study, adding PRGF to DBBM enhanced osteogenesis in rabbit calvarias. Applying autologous fibrin membrane in the defects was not helpful.