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.

Advances in Platelet Rich Plasma-Derived Extracellular Vesicles for Regenerative Medicine: A Systematic-Narrative Review.

The use of platelet-rich plasma (PRP) has gained increasing interest in recent decades. The platelet secretome contains a multitude of growth factors, cytokines, chemokines, and other biological biomolecules. In recent years, developments in the field of platelets have led to new insights, and attention has been focused on the platelets' released extracellular vesicles (EVs) and their role in intercellular communication. In this context, the aim of this review was to compile the current evidence on PRP-derived extracellular vesicles to identify the advantages and limitations fortheir use in the upcoming clinical applications. A total of 172 articles were identified during the systematic literature search through two databases (PubMed and Web of Science). Twenty publications met the inclusion criteria and were included in this review. According to the results, the use of PRP-EVs in the clinic is an emerging field of great interest that represents a promising therapeutic option, as their efficacy has been demonstrated in the majority of fields of applications included in this review. However, the lack of standardization along the procedures in both the field of PRP and the EVs makes it extremely challenging to compare results among studies. Establishing standardized conditions to ensure optimized and detailed protocols and define parameters such as the dose or the EV origin is therefore urgent. Further studies to elucidate the real contribution of EVs to PRP in terms of composition and functionality should also be performed. Nevertheless, research on the field provides promising results and a novel basis to deal with the regenerative medicine and drug delivery fields in the future.

The Dual-Responsive Interaction of Particulated Hyaline Cartilage and Plasma Rich in Growth Factors (PRGF) in the Repair of Cartilage Defects: An In Vitro Study.

The treatment of chondral and osteochondral defects is challenging. These types of lesions are painful and progress to osteoarthritis over time. Tissue engineering offers tools to address this unmet medical need. The use of an autologous cartilage construct consisting of hyaline cartilage chips embedded in plasma rich in growth factors (PRGF) has been proposed as a therapeutic alternative. The purpose of this study was to dig into the potential mechanisms behind the in vitro remodelling process that might explain the clinical success of this technique and facilitate its optimisation. Chondrocyte viability and cellular behaviour over eight weeks of in vitro culture, type II collagen synthesis, the dual delivery of growth factors by hyaline cartilage and PRGF matrix, and the ultrastructure of the construct and its remodelling were characterised. The main finding of this research is that the cartilage fragments embedded in the three-dimensional PRGF scaffold contain viable chondrocytes that are able to migrate into the fibrin network, proliferate and synthesise extracellular matrix after the second week of in vitro culture. The characterization of this three-dimensional matrix is key to unravelling the molecular kinetics responsible for its efficacy.

PRGF Membrane with Tailored Optical Properties Preserves the Cytoprotective Effect of Plasma Rich in Growth Factors: In Vitro Model of Retinal Pigment Epithelial Cells.

The present study evaluates the ability of a novel plasma rich in growth factors (PRGF) membrane with improved optical properties to reduce oxidative stress in retinal pigment epithelial cells (ARPE-19 cells) exposed to blue light. PRGF was obtained from three healthy donors and divided into four main groups: (i) PRGF membrane (M-PRGF), (ii) PRGF supernatant (S-PRGF), (iii) platelet-poor plasma (PPP) membrane diluted 50% with S-PRGF (M-PPP 50%), and (iv) M-PPP 50% supernatant (S-PPP 50%). ARPE-19 cells were exposed to blue light and then incubated with the different PRGF-derived formulations or control for 24 and 48 h under blue light exposure. Mitochondrial and cell viability, reactive oxygen species (ROS) production, and heme oxygenase-1 (HO-1) and ZO-1 expression were evaluated. Mitochondrial viability and cell survival were significantly increased after treatment with the different PRGF-derived formulations. ROS synthesis and HO-1 expression were significantly reduced after cell treatment with any of the PRGF-derived formulations. Furthermore, the different PRGF-derived formulations significantly increased ZO-1 expression in ARPE-19 exposed to blue light. The new PRGF membrane with improved optical properties and its supernatant (M-PPP 50% and S-PPP 50%) protected and reversed blue light-induced oxidative stress in ARPE-19 cells at levels like those of a natural PRGF membrane and its supernatant.

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.

Development of a new plasma rich in growth factors membrane with improved optical properties.

PURPOSE: The aim of the present work was to develop a fibrin membrane using plasma rich in growth factors (PRGF) technology with improved optical properties to be used for the treatment of ocular surface diseases. BASIC PROCEDURES: Blood was drawn from three healthy donors, and the volume of PRGF obtained from each donor was divided into two main groups: i) PRGF or ii) platelet-poor plasma (PPP). Each membrane was then used pure or diluted to 90 %, 80 %, 70 %, 60 % and 50 %. The transparency of each of the different membranes was evaluated. The degradation and morphological characterization of each membrane was also performed. Finally, a stability study of the different fibrin membranes was performed. MAIN FINDINGS: The transmittance test showed that the fibrin membrane with the best optical characteristics was obtained after removal of platelets and dilution of fibrin to 50 % (50 % PPP). No significant differences (p > 0.05) were observed between the different membranes in the fibrin degradation test. The stability test showed that the membrane at 50 % PPP retains its optical and physical characteristics after storage at - 20 °C for 1 month compared to storage at 4 °C. PRINCIPAL CONCLUSIONS: The present study describes the development and characterization of a new fibrin membrane with improved optical characteristics while maintaining mechanical and biological characteristics. The physical and mechanical properties of the newly developed membrane are preserved after storage for at least 1 month at - 20 °C.

On Peri-Implant Bone Loss Theories: Trying To Piece Together the Jigsaw.

This review aims to explore the plausibility of new theories on the etiopathogenesis of marginal bone loss (MBL) and peri-implantitis (PI) and to discuss possible underlying pathogenic mechanisms. The former concept of osteointegration of dental implants can now be conceptualized as a foreign body response histologically characterized by a bony demarcation in combination with chronic inflammation. Different risk factors can provoke additional inflammation and, therefore, pro-inflammatory cytokine release in soft tissues and bone, leading to an overpass of the threshold of peri-implant bone defensive and regenerative capacity. Progressive bone loss observed in MBL and PI is ultimately due to a localized imbalance in the receptor activator of nuclear factor kappaB ligand (RANKL)/Receptor activator of nuclear factor κ B (RANK)/osteoprotegerin (OPG) pathway in favor of increased catabolic activity. The genetic background and the severity and duration of the risk factors could explain differences between individuals in the threshold needed to reach an imbalanced scenario. MBL and PI pathogenesis could be better explained by the "inflammation-immunological balance" theory rather than a solely "infectious disease" conception. The link between the effect of biofilm and other risk factors leading to an imbalanced foreign body response lies in osteoclast differentiation and activation pathways (over)stimulation.

Performance of Immediately Loaded Short Implants Using Low-Speed Drilling Protocol and Plasma Rich in Growth Factors: A Controlled Retrospective Observational Study.

Short dental implants are an alternative to surgical bone augmentation procedures and the placement of longer implants. The high predictability of short implants has encouraged clinicians to load them immediately. However, there are few studies assessing the influence of immediate vs delayed loading of short (< 8 mm) implants. The purpose of this retrospective study was to report the mid-term (5-year) outcomes (survival and marginal bone loss [MBL]) of immediate vs delayed loading of short implants. A total of 44 patients with 149 short implants fulfilled the inclusion criteria (95 and 54 implants with delayed and immediate loading, respectively). During the follow-up period, descriptive clinical variables, implant survival, MBL, and prosthetic complications were recorded and statistically analyzed. The mean follow-up time was 60 ± 40 months. The overall cumulative implant survival was 95.6%, and MBL was -0.1 ± 0.7 mm. No statistically significant differences were detected between the immediate and delayed loading groups in terms of implant survival (92.6% vs 97.5%) or MBL (-0.2 ± 0.8 mm vs -0.1 ± 0.7 mm), respectively. According to the results of this study, the immediate loading of short implants demonstrated predictability at the mid-term followup time. These results must be confirmed in future prospective studies. Int J Periodontics Restorative Dent 2023;43:233-239. doi: 10.11607/prd.5203.

A Novel Technique for Preparation of Recipient Site and Autologous Bone Grafting in Autotransplantation of Single-Rooted Teeth: A Report of Two Cases.

Low-speed drilling without irrigation has a long history of use in implant dentistry. It provides the advantage of avoiding the wash-out of proteins and biomolecules from the bone. In this case report, we describe the novel use of this drilling protocol in the preparation of bone alveolus during the procedure of tooth autotransplantation. Two cases with early tooth loss in the upper maxillary arch were treated by the autotransplantation of permanent teeth with immature root development and the use of plasma rich in growth factors. Autologous bone fragments (from drilling) were harvested and used for alveolar bone augmentation. The follow-up time was three and seven years since tooth autotransplantation. All the autotransplanted teeth achieved a closed apex with no signs of loss of vitality. Low-speed drilling without irrigation did not jeopardize the outcomes of tooth autotransplantation and warrants further investigation in the context of periodontal ligament healing.

The effect of mindfulness on the inflammatory, psychological and biomechanical domains of adult patients with low back pain: A randomized controlled clinical trial.

OBJECTIVE: This study aims to study the effect of mindfulness-based program on the psychological, biomechanical and inflammatory domains of patients with chronic low back pain. METHODS: A multicentre randomized and controlled clinical trial of parallel groups in patients with chronic low back pain between March 2019 to March 2020. Participants with no experience in mindfulness based intervention, were randomized to receive (36 patients) or not (34 patients) mindfulness-based stress reduction program for chronic back pain (MBSR-CBP). The program was performed in 9 sessions. Patients with chronic low back pain due to symptomatic discopathy (degenerative disc disease or herniated disc) were included. The principal outcome was changes in the blood level of cortisol and cytokines (tumor necrosis factor- α (TNF- α), interleukin-1ß (IL-1ß), interleukin-6 (IL-6) and interleukin-17 (IL-17)). Secondary outcomes (psychological factors, pain, and quality of life) were measured by validated questionnaires. RESULTS: Of the 96 randomized patients, 70 who completed the study were included in the analysis (mean [range] age: 53 [33-73] years; 66% females). MBSR-CBP stopped the increase in cortisol, and reduced pro-inflammatory cytokine IL-1ß (p = 0.05). It reduced depression (p = 0.046) and stress (p = 0.0438), perceived pain (p < 0.0001), and limitations related to health (p < 0.0001). It also increased the physical function (p = 0.002) and sleep quality (p = 0.05). Furthermore, it significantly increased life satisfaction (0.006), well-being (p = 0.001) and vitality (p < 0.0001). It also increased self-compassion (p < 0.0001) and significantly reduced the overidentification (p<0.0001) and catastrophization (p = 0.002). CONCLUSIONS: MBSR-CBP could be part of a multidisciplinary approach in the management of patients suffering from chronic low back pain.

Platelet-Rich Plasma as an Alternative to Xenogeneic Sera in Cell-Based Therapies: A Need for Standardization.

There has been an explosion in scientific interest in using human-platelet-rich plasma (PRP) as a substitute of xenogeneic sera in cell-based therapies. However, there is a need to create standardization in this field. This systematic review is based on literature searches in PubMed and Web of Science databases until June 2021. Forty-one studies completed the selection criteria. The composition of PRP was completely reported in less than 30% of the studies. PRP has been used as PRP-derived supernatant or non-activated PRP. Two ranges could be identified for platelet concentration, the first between 0.14 × 106 and 0.80 × 106 platelets/µL and the second between 1.086 × 106 and 10 × 106 platelets/µL. Several studies have pooled PRP with a pool size varying from four to nine donors. The optimal dose for the PRP or PRP supernatant is 10%. PRP or PRP-derived supernatants a have positive effect on MSC colony number and size, cell proliferation, cell differentiation and genetic stability. The use of leukocyte-depleted PRP has been demonstrated to be a feasible alternative to xenogeneic sera. However, there is a need to improve the description of the PRP preparation methodology as well as its composition. Several items are identified and reported to create guidelines for future research.

Short Narrow Dental Implants versus Long Narrow Dental Implants in Fixed Prostheses: A Prospective Clinical Study.

There is a paucity of studies that assess short and narrow dental implants. This prospective study aimed to evaluate the performance of both short (≤8 mm) and narrow (≤3.5 mm width) dental implants supporting fixed prostheses in the atrophic maxilla or mandible. Towards that aim, patients with short implants were included in the study. The control group was those with long and narrow dental implants (length > 8 mm and diameter ≤ 3.5 mm). Clinical and demographic variables were extracted from clinical records. During the follow-up, implant survival and marginal bone loss were evaluated and statistically analysed. Forty-one implants were included (18 and 23 implants in the test and control groups, respectively). The median follow-up time was 26 months since insertion in both groups. The results revealed that there was no implant failure and no statistically significant differences in terms of marginal bone loss. Only one screw-loosening effect occurred in the short implants group. Short, narrow dental implants could be an alternative for the restoration of severely resorbed jaws.

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.

Oral Pemphigoid Recalcitrant Lesion Treated with PRGF Infiltration. Case Report.

Mucous membrane pemphigoid (MMP) is a heterogeneous group of chronic autoimmune subepithelial blistering diseases. Oral involvement is present in almost all patients, may represent the onset of the disease, and causes different degrees of pain, dysphagia, soreness, and bleeding. Treatment is based on systemic and/or oral corticoids, or other immunosuppressants. Occasionally, oral lesions can show a poor response to standard treatments. We present the case of a 61-year-old female patient with a painful extensive MMP oral ulcerative lesion recalcitrant to previous systemic azathioprine and local triamcinolone treatment, which was successfully treated in a novel way using PRGF infiltrations as adjuvant. After four weekly infiltrations, pain was reduced from 10 to 0 in a VAS and the lesion was completely healed. The patient continued with a low dose maintenance immunosuppressive treatment (prednisone 5 mg/day PO), and after 13 months of follow-up, there was no relapse of the lesion and no side effects. Although future research is necessary to confirm these observations, PRGF could be a useful adjuvant for the management of extensive mucous membrane pemphigoid oral lesions.

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.

Platelet rich plasma in oral and maxillofacial surgery from the perspective of composition.

The application of platelet-rich plasma (PRP) in oral and maxillofacial surgery has been thoroughly studied in the last two decades. Currently, different types of PRP are applied in the clinical practice, being the presence or absence of leukocytes one of the classification criteria. However, there is poor evidence assessing the influence of the PRP composition in their efficacy. In this context, the aim of this narrative review is to compile the existing evidence covering the efficacy of PRP in oral and maxillofacial surgery, starting from a systematic literature search and to qualitatively describe the efficacy outcomes from the composition perspective. According to the results of this review, the application of PRP in oral and maxillofacial surgery is a potential strategy to improve soft- and hard-tissue regeneration, observing differences in the efficacy of PRP depending on its composition and the studied application. P-PRP (the absence of leukocytes) has been more consistent in achieving beneficial effects in alveolar ridge preservation, management of post-extraction complications, bone augmentation and temporomandibular joint disorders. For that, the composition and methodology used to prepare the PRP should be a critical point when evaluating the efficacy of PRP.

Performance of the counter-torque technique in the explantation of nonmobile dental implants.

BACKGROUND: The application of the counter-torque technique has been proposed as a conservative and atraumatic alternative for the explantation of nonmobile dental implants. The objective of this report is to assess the performance of this technique in a large number of patients. RESULTS: Three hundred and fifty-five patients were treated for the explantation of 749 nonmobile dental implants. The explantations were performed by the application of counter-torque to break the bone-implant interface. Successful implant explantation was achieved in 98.4% of the implants. The frequency of complications was 1.3%, most commonly related to the appearance of fissure lines at the implant neck. CONCLUSIONS: The counter-torque technique has a high success rate but is not exempt from complications, although at a very low rate.