Material-dependent effects of injectable platelet rich-fibrin on growth factor release, inflammation, and osteoclast activity: an in vitro study.

Context: Platelet concentrates are rich in growth factors that assist in regenerative medicine to promote healing and tissue regeneration. Similarly, partially demineralized tooth is a storehouse of many growth factors, assisting in bone regeneration. Hence, the present study aimed to quantify the release of growth factors from different platelet concentrates individually and when mixed with a partially demineralized tooth matrix. Method: Human growth factors such as bFGF, EGF, PDGF-AB, TGF-beta-1, and VEGF-A present in platelet-rich fibrin and injectable platelet-rich fibrin from young and old male and female donors were quantified separately. Then these platelet concentrates were then mixed with a partially demineralized tooth matrix, which was powdered using a Smart Dentin Grinder. An enzyme-linked immunosorbent assay was used for the quantification of growth factors. Results: The release of growth factors, such as platelet-rich fibrin and injectable platelet-rich fibrin, was not statistically significant; however, it was significantly greater when i-PRF was mixed with a partially demineralized tooth matrix. Conclusion: The study revealed that the expression of growth factors was significantly greater when a partially demineralized tooth matrix was mixed with injectable platelet-rich fibrin than when combined with platelet-rich fibrin.

ObjectiveThe release of growth factors in injectable platelet-rich fibrin (I-PRF) exhibits a peak within 24 hours and subsequent decline by day 10, underscoring immediate application, limiting its effectiveness in alveolar bone repair. In order to enhance its regenerative potential, I-PRF can be combined with biomaterial scaffolds such as collagen-chitosan hydrogels, which mimic the extracellular matrix and support tissue regeneration. This combination has been shown to enhance cellular signaling and tissue repair. This study aimed to analyze the characterization of collagen-chitosan hydrogels with I-PRF and determine the growth factor release pattern that occurs after mixing.Materials and MethodsCollagen-chitosan hydrogels were prepared and combined with I-PRF at a 1:1 ratio. The structural characterization of these hydrogels, both with and without I-PRF, was performed using Fourier transform infrared spectroscopy (FTIR), enabling the comparison of absorption bands. Furthermore, the release profiles of transforming growth factor-beta 1 (TGF-β1) and platelet-derived growth factor AB (PDGF-AB) were assessed in two experimental groups: The first group consisted of I-PRF alone, while the second group comprised of I-PRF combined with collagen-chitosan hydrogels. Growth factor release was evaluated at multiple time points (days 1, 3, 5, 7, 9, 11, 13, 15, and 17) using enzyme-linked immunosorbent assay. The resulting absorbance values were converted into concentration measurements (pg/mL) using a standard calibration curve. Statistical analysis was conducted using two-way analysis of variance followed by apost hocleast significant difference test.ResultsFTIR analysis demonstrated that the functional groups present in the collagen-chitosan hydrogel remained unchanged following the incorporation of I-PRF, confirming the formation of physical rather than chemical bonds. Subsequent analysis revealed statistically significant differences in the release patterns of TGF-β1 and PDGF-AB between the two groups (p < 0.05). The combination of collagen-chitosan hydrogel and I-PRF exhibited a more stable and sustained release profile from day 1 to day 17.ConclusionThe combination of I-PRF with collagen-chitosan hydrogels does not alter the fundamental chemical structure of the scaffold. However, this combination does influence the controlled release of growth factors. This finding indicates that the synergistic interaction between collagen and chitosan enhances the hydrogel's properties, suggesting its potential as a promising biomaterial for use as a scaffold in bone regeneration.

This study examined the potential of a new porous calcium hydroxyapatite scaffold covered with poly (lactide-co-glycolide) (PLGA) as a bone substitute, identifying its advantages over Geistlich Bio-Oss®, considered the gold standard, in in vivo biofunctionality investigations. Structural and morphological properties of the new scaffold were analyzed by scanning electron and atomic force microscopy. The biofunctionality assays were performed on New Zealand white rabbits using new scaffold for filling full-thickness defects of critical size. The evaluated parameters were: the presence of macrophages, giant cells, monoocytes, plasma cells, granulocytes, neoangiogenesis, fibroplasia, and the percentage of mineralization. Parallel biofunctionality assays were performed using Geistlich Bio-Oss®. The appearance of bone defects 12 weeks after the new scaffold implantation showed the presence of a small number of typical immune response cells. Furthermore, significantly reduced number of capillary buds, low intensity of fibroplasia and high degree of mineralization in a lamellar pattern indicated that the inflammation process has been almost completely overcome and that the new bone formed was in the final phase of remodeling. All biofunctionality assays proved the new scaffold's suitability as a bone substitute for applications in maxillofacial surgery. It showed numerous biological advantages over Geistlich Bio-Oss® which was reflected mainly as a lower number of giant cells surrounding implanted material and higher degree of mineralization in new formed bone.

Background: Periodontal regeneration in particular represents regain of lost periodontal tissues either by regenerative techniques to be used alone or in combination with an adjunct. Platelet-rich Fibrin (PRF) is an autologous product that is derived from whole blood by applying the process of gradient density centrifugation. Use of platelet concentrates is promising due to its autologous nature, release of multiple growth factors, low cost, ease of procurement and comfortable handling. Injectable PRF (i-PRF) can be combined with bone grafts to form sticky bone with superior handling and healing properties. Hereby, we report a case with intra bony defects treated with sticky bone and barrier membrane with a 1-year follow-up. Methodology: 10 ml of the patient?s venous blood was collected and i-PRF was obtained by low centrifugation protocol. Sticky bone was prepared by mixing i-PRF with alloplast and was placed in intrabony defect. A bioresorbable collagen membrane was placed over the sticky bone followed by periodontal flap closure. Results: A reduction in the Probing pocket depth (PPD) from 7mm (Pre-operative) to 3mm, Clinical attachment loss (CAL) from 8mm to 4mm. Conclusion: Significant improvement in clinical parameters such as PPD, CAL and radiographic bone fill indicates success of regenerative therapy using i-PRF with bone grafts.

Platelet concentrates have been used in various applications within Oral and Maxillofacial Surgery and Implantology in order to promote tissue repair. The Platelet-Rich Plasma (PRP) is the first generation of platelet concentrates and recent studies question its actual effectiveness on tissue healing due to the rapid release of growth factors. The second generation of platelet concentrates, the Platelet-Rich Fibrin (PRF), has peculiar features in the release of growth factors, which occurs slowly and gradually and thus seems to promote a more promising effect over the tissues under repair in comparison with PRP, especially regarding to bone. However, the literature data are different and better results seem to be obtained when the PRF is mainly used in combination with other graft materials. Additional well-designed studies are needed to better clarify this matter.

Introduction: Chronic lung disorders involve pathological alterations in the lung tissue with hypoxia as a consequence. Hypoxia may influence the release of inflammatory mediators and growth factors including vascular endothelial growth factor (VEGF) and prostaglandin (PG)E2. The aim of this work was to investigate how hypoxia affects human lung epithelial cells in combination with profibrotic stimuli and its correlation to pathogenesis. Methods: Human bronchial (BEAS-2B) and alveolar (hAELVi) epithelial cells were exposed to either hypoxia (1% O2) or normoxia (21% O2) during 24h, with or without transforming growth factor (TGF)-β1. mRNA expression of genes and proteins related to disease pathology were analysed with qPCR, ELISA or immunocytochemistry. Alterations in cell viability and metabolic activity were determined. Results: In BEAS-2B and hAELVi, hypoxia significantly dowregulated genes related to fibrosis, mitochondrial stress, oxidative stress, apoptosis and inflammation whereas VEGF receptor 2 increased. Hypoxia increased the expression of Tenascin-C, whereas both hypoxia and TGF-β1 stimuli increased the release of VEGF, IL-6, IL-8 and MCP-1 in BEAS-2B. In hAELVi, hypoxia reduced the release of fibroblast growth factor, epidermal growth factor, PGE2, IL-6 and IL-8, whereas TGF-β1 stimulus significantly increased the release of PGE2 and IL-6. TGF-β1 stimulated BEAS-2B cells showed a decreased release of VEGF-A and IL-8, while TGF-β1 stimulated hAELVi cells showed a decreased release of PGE2 and IL-8 during hypoxia compared to normoxia. Metabolic activity was significantly increased by hypoxia in both epithelial cell types. Discussion: In conclusion, our data indicate that bronchial and alveolar epithelial cells respond differently to hypoxia and profibrotic stimuli. The bronchial epithelium appears more responsive to changes in oxygen levels and remodelling processes compared to the alveoli, suggesting that hypoxia may be a driver of pathogenesis in chronic lung disorders.

My group is interested in new technologies and strategies for osteochondral tissue regeneration. Some of the core tenets of our philosophy for tissue regeneration include the use of “raw materials” as building blocks, the engineering of continuous gradients, and leveraging osteochondral tissue engineering to enhance cartilage regeneration. The use of scaffolds built exclusively from microspheres provides a means for both raw materials and gradients to be employed in osteochondral tissue engineering. With the use of microspheres, we have shown that a single osteochondral biomaterial implant can be created with a continuous gradient in both material composition and growth factor release, with a seamless transition from one side to the other. A series of in vivo tests in both the knee and in the temporomandibular joint (TMJ, or jaw joint) have yielded promising results for the use of microsphere‐based scaffolds. As an alternate strategy, we are pioneering the use of interpenetrating network (IPN) hydrogels to encapsulate cells for cartilage tissue engineering. We have shown that two different hydrogel networks can be combined in such a way to create an IPN hydrogel with mechanical integrity far superior to either of the original gels, with viable cells encapsulated. This IPN approach has been further enhanced with the incorporation of “raw materials” for cartilage.

To: Editor, The Angle OrthodontistRe: Response To: Effect of injectable platelet-rich fibrin (i-PRF) on the rate of tooth movement. Emire Aybuke Erdur, Kotter Karakaslı, Elif Oncu, Bahadir Ozturk, Sema Hakkı. Angle Orthod. 2021;91:285-292.Thank you for giving us the chance to respond to Dr.Tripathi and colleagues' comments about our article. These comments enable further clarification regarding some aspects of our research.There was no clinical study about i-PRF to refer to when designing this study. To reduce treatment time, many techniques have been described in the literature to accelerate tooth movement based on the regional acceleratory phenomenon.1–5 The procedures applied in these studies were in the direction of tooth movement. Also, in an animal study about PRP, 2 the injection was applied to the direction of tooth movement. For this reason, we applied i-PRF only on distal aspect of the canine. The amount of i-PRF was standardized as 4 mL and was injected intraligamentally into the distobuccal and distopalatal side of the canine tooth (2 mL for each side). An in vivo study showed that a new formulation of PRF (A-PRF, i-PRF) had a gradual release of growth factors, up to about a week, and stimulated significantly greater growth factor release over time.6 So it should have been longer than a week for the second activation. In our study, the study group received i-PRF two times: just after premolar extraction and at the second week of distalization. The contralateral side served as a control and received only a sham injection. The first PRF application and extraction of the teeth was accomplished at the same time. We mentioned the closure of the space in the discussion section as i-PRF demonstrated a significant increase in the rate of canine tooth movement. The positive effect of i-PRF on the rate of tooth movement started in the first week and was seen throughout the follow-up period.The study group received i-PRF two times: just after premolar extraction and at the second week of distalization. We mentioned before that i-PRF had a gradual release of growth factors, up to about 1 week, and stimulated significantly higher growth factor release. The aim in our study was to determine whether the effect of i-PRF on tooth movement would continue in the long term. To determine this, we evaluated tooth movement velocity and the correlation at T3 and T4 time points. The results were presented in Table 1 in the study.7We mentioned in the discussion section that OPG inhibits osteoclast differentiation by binding to RANKL. These cytokines play significant roles in reinforcing and activating osteoclast precursor cells. Increased release of these factors is accompanied by higher osteoclast activation and therefore a higher rate of tooth movement.1,3 Periodontal ligament cells can regulate osteoclastogenesis by reverse mechanisms with stimulation of resorptive activity by RANKL and inhibition by OPG. Kanzaki et al.8.9 demonstrated that compressive force increased the production of RANKL and decreased that of OPG in human periodontal ligament cells. Baloul et al.3 reported that alveolar decortication stimulated the RANKL/OPG ratio where an increase in RANKL was associated with decreased OPG which was more rapid and simultaneous compared to conventional tooth movement.There are many studies that have had similar results; a low OPG level may be related to rapid orthodontic tooth movement. There are many factors that affect the difference in study results. Only ten adolescent patients were included in the study you mentioned. GCF was collected at the distal cervical margins of the experimental and control teeth at 0, 1, 24, and 168 hours after the force was applied. The statistical analysis was performed using a non-parametric test (Mann-Whitney U-test). Also, the researchers had attributed this difference in RANKL and OPG to their experimental design, which did not provide for a continuous and consistent force over the entire experimental period of 168 h.

The ability of polymorphonuclear leukocytes (PMNs) to modulate endothelial cell (EC) activation was investigated. Adding PMNs to cultured HUVECs resulted in a release of IL-6 (888 +/- 71 pg/ml, a 35-fold increase over release by the two cell types alone) and IL-8 (45.2 +/- 14.5 ng/ml, a 6.4-fold over PMN release alone and a 173-fold increase over EC release alone). In contrast, the release of TNF-alpha, IL-1beta, and platelet-derived growth factor was not affected by the EC-PMN coculture. Neutralizing mAbs to ICAM-1 or beta2 integrins or a physical segregation of PMNs and ECs did not reduce EC stimulation. In contrast, cell-free supernatants of PMNs recapitulated EC activation with an 18-fold up-regulation of EC IL-6 mRNA. The filtration of PMN supernatant or PMN pretreatment with metabolic antagonists or membrane cross-linking agents all suppressed EC activation. By flow cytometry, PMNs released in the supernatant, heterogeneous membrane-derived microparticles containing discrete proteins of 28 to 250 kDa as resolved by SDS-PAGE. PMN microparticle formation was enhanced by inflammatory stimuli, including formyl peptide and phorbol ester, and was time-dependent, reaching a plateau after a 1-h incubation from stimulation. Purified PMN microparticles induced EC IL-6 release in a reaction that was quantitatively indistinguishable from that observed with unfractionated PMN supernatant and unaffected by a neutralizing Ab to soluble IL-6R. These findings demonstrate that membrane microparticles released from stimulated PMNs are competent inflammatory mediators to produce EC activation and cytokine gene induction.

Considerable progress has been made in the development and testing of agents to treat osteoporosis. Most impressive are reports on new antiresorptive agents--both bisphosphonates (ibandronate and zoledronic acid) and monoclonal antibodies (MAbs) (denosumab) directed against receptor activator of nuclear factor kappaB-ligand, a key molecule in the control of commitment and activation of osteoclasts. Bisphosphonates promise convenience and potency at slowing bone loss, whereas denosumab offers powerful suppression of resorption and rapid offset of action. Attention is also shifting from the osteoclast as a target for new therapies to the osteoblast and the osteocyte, with its complex network within the depths of bone. Wnt signaling through the frizzled receptor and its coreceptor, the low-density lipoprotein receptor related protein-5, appears from both molecular and in vivo evidence to be a pivotal pathway for modulating osteoblastic activity, bone formation, and bone strength. The recently identified product of the SOST gene or sclerostin has also been shown to block Wnt signaling. Sclerostin is produced by the osteocytes buried in the bone and is a new target to treat bone loss. Clinical trial reports indicate that the calcimimetic cinacalcet can effectively treat PTH hypersecretion due to primary and secondary hyperparathyroidism and parathyroid carcinoma. Lastly, it is now recognized that the matrix protein dentin matrix protein-1 enhances the release of the phosphate-regulating factor fibroblast growth factor 23 and that mutations in dentin matrix protein-1 play a causative role in a form of hypophosphatemic rickets.

Tumor cells can secrete various cytokines which can enhance the activity of osteoclast in the bone microenvironment, and osteoclast can promote the release of many growth factors buried in bone matrix which would promote the growth and invasion of tumor cells. Thus, a ' vicious cycle' of bone destruction is developed in the bone metastatic microenvironment. Bone metastatic microenvironment facilitate this ' vicious cycle', while it also provides potential targets for the treatment of bone metastases. Osteoprotegerin, receptor activator of nuclear factor-κB and its ligand system are the typicality of molecular targets. Bone metastasis can promote the secretion of RANKL and the expression of OPG. The disbalance of RANKL/OPG is an important inducing factor for bone destruction. Many studies have shown that transforming growth factor-β which is produced by osteoclast plays an important role in mediating ' vicious cycle'. Src family tyrosine kinase, endothelin A receptor, matrix metalloproteinase, and cathepsin K are the potential targets of bone metastasis. Pharmacologic agents such as denosumab, can inhibit the ' vicious cycle' of bone metastasis. In addition to suppress bone destruction by Pharmacologic agents, they also can produce direct antitumor effect. They can delay the occurrence of skeletal related events, prolong the overall survival, and play an important role in patient's quality of life at last. Patients with bone metastasis have already benefited from systemic molecular targeted therapies, and further researches would be of great importance in improving patient therapeutically selections and enhancing the effect.

Normal bone remodeling maintains an appropriate balance between the action of osteoclasts (bone-resorbing cells) and osteoblasts (bone-forming cells). Skeletal malignancies, including bone metastases, disrupt the OPG-RANKL-RANK signal transduction pathway and promote enhanced osteoclast formation, thereby accelerating bone resorption and inducing bone loss. This osteolysis in turn leads to the release of bone-derived growth factors, contributing to a “vicious cycle” in which interactions between tumor cells and osteoclasts not only lead to increased osteoclastogenesis and osteolytic activity, but also aggressive growth and behavior of the tumor cells. The osteolytic complications associated with bone metastases are caused by tumor-induced alterations of the OPG-RANKL-RANK system, which are accompanied by enhanced bone resorption and disassociated from counterbalancing bone formation by osteoblasts.

Material composition and porous structure are important factors in the formation and maturation of newly formed bone and replacement of materials by new bone. Conventional bone graft materials often lack suitability for bone generation because of the complexity of their macroporous structures, which can interfere with the penetration of cells related to bone remodeling and angiogenesis in the materials. In the present study, carbonate apatite (CO3Ap), hydroxyapatite (HAp), and β-tricalcium phosphate (TCP) honeycomb granules (HCGs) with uniformly sized macropores (∼115 μm) were fabricated. These HCG macropores were arranged in a regular fashion and penetrated straight into the granules. They were implanted into a rabbit femur defect for further evaluation. In the CO3Ap HCG implantation group, mature bone formed within CO3Ap HCG macropores by 4 weeks after grafting, and a large portion of CO3Ap HCGs was replaced by new bone at 12 weeks. By contrast, in the β-TCP HCG implantation group, new bone was not always formed in the regions after β-TCP HCG disappearance, and immature bone was present within β-TCP HCG macropores even after 12 weeks. HAp HCGs were not resorbed, and their macropores were filled with immature bone. The area of mature bone in the CO3Ap HCG implantation group was 3.3 and 1.6 times higher at 4 weeks and 2.2 and 1.7 times higher at 12 weeks compared with the HAp and β-TCP HCG implantation groups, respectively. Furthermore, the degrees of bone maturation for CO3Ap, HAp, and β-TCP HCGs were 100, 34, and 64% at 4 weeks, and 100, 54, and 69% at 12 weeks, respectively. Thus, the composition of the HCGs affected bone formation and maturation.

β-tricalcium phosphate (β-TCP) is a widely utilized resorbable bone graft material, whose surface charge can be modified by electrical polarization. However, the specific effects of such a charge modification on osteoblast and osteoclast functions remain insufficiently studied. In this work, electrically polarized β-TCP with a high surface charge density was synthesized and evaluated in vitro in terms of its physicochemical properties and biological activity. Polarization was performed to achieve a high surface charge density, which was quantified using a thermally stimulated depolarization current. The proliferation and differentiation of MC3T3-E1 osteoblast-like cells were assessed via WST-8 and alkaline phosphatase assays. Tartrate-resistant acid phosphatase (TRAP) activity and a resorption pit assay were used to evaluate the impact of surface charge on RAW264.7 osteoclast-like cell activity. Polarized β-TCP exhibited a surface charge of 1.3 mC cm-2. Electrically polarized surfaces significantly enhanced osteoblast proliferation and differentiation. TRAP activity assays demonstrated effective osteoclast differentiation of RAW264.7 cells, with enhanced activity observed on charged surfaces. Resorption pit assays further revealed improved osteoclast resorption capacity on β-TCP surfaces with a polarized charge. These findings indicate that β-TCP with a highly dense surface charge promotes osteoblast proliferation and differentiation, as well as osteoclast activity and resorption capacity.

Constitutive Release of Cytokines by Human Oral Keratinocytes in an Organotypic Culture