REGENERACINIŲ ENDODONTINIŲ PROCEDŪRŲ VEIKSMINGUMO PALYGINIMAS, GYDANT NESUSIFORMAVUSIŲ NUOLATINIŲ DANTŲ ŠAKNŲ PULPOS NEKROZĘ

Šiuolaikinės regeneracinės endodontinės procedūros skatina naujo vaskuliarizuoto audinio susidarymą kanalo ertmėje, todėl galima tikėtis tolimesnio dantų šaknų formavimosi ir viršūninio periodontito sugijimo. Stebint tokius klinikinius bei rentgenologinius pokyčius, regeneracinės endodontinės procedūros gali būti svarstytinos kaip pirmo pasirinkimo gydymo priemonė, nuolatinių dantų nesusiformavusiose šaknyse išsivysčius pulpos nekrozei. Darbo tikslas – atlikti mokslinės literatūros analizę ir palyginti regeneracines endodontines procedūras taikant PRF, PRP, PP arba kraujo krešulį, kai gydomi nuolatiniai dantys, esant nevisiškai susiformavusioms šaknims ir pulpos nekrozei. Metodika. Atlikta elektroninė literatūros paieška anglų kalba PubMed duomenų bazėje, naudojant šias raktažodžių kombinacijas: tooth regeneration, platelet-rich plasma, platelet-rich fibrin, platelet pellet, scaffold, immature teeth. Rezultatai. Statistiškai reikšmingas danties šaknies pailgėjimas, atlikus kontrolę po vienerių metų su kraujo krešuliu, nustatytas dviejuose tyrimuose, o naudojant PRP – viename. Išvados. Atliekant regeneracines endodontines procedūras, svarbu sukurti natūralų biologinį audinio regeneravimo matriksą, kuris gali būti PRF, PRP, PP ar kraujo krešulio pavidalu. Nėra patvirtintų mokslinių įrodymų, kad danties kanale vietiškai naudojami antibiotikai teigiamai veiktų regeneracines dantų procedūras. EDTA naudojimas nerekomenduojamas.

How Rich is Platelet Rich Plasma? A Simple Preparation Method of PRP and Its Quality According to Various Classification Systems

Background: platelets are a rich source of various growth factors and hence platelet rich plasma (PRP) is being used therapeutically in the fi eld of dermatology, orthopaedics and dentistry with promising results. However, methods of preparation of PRP vary with many commercial kits also available. Scoring systems like DEPA score, PAW system and sports medicine criteria score help in determining the quality of the fi nal product and guide therapy. Here we test a simple two step method for preparation of PRP and score it according to the abovementioned criteria while comparing it with the commercial kits analysed in literature. Methods: 10 ml whole blood was collected in 1.5 ml ACD solution from 100 healthy willing participants and analysed for platelet concentration. Sample was then centrifuged at 1600 rpm X 4 minutes in a calibrated laboratory centrifuge and supernatant was collected in a sterile tube using a sterile syringe. The supernatant was further concentrated for platelets by centrifuging at 3600 rpm X 10 minutes to form a platelet pellet at the bottom. All but 1 ml supernatant (Platelet defi cient plasma) was discarded and the platelet pellet was suspended again to form platelet rich plasma. Platelet count was estimated in this sample as done with the whole blood sample. The results were compared and analysed using statistical methods to determine the effi cacy of concentrating platelets by this method. Results: The tested method gave a fi nal concentration of more than four times the baseline concentration with approximately 30% effi ciency of capture and 80% purity. This was comparable to majority of the commercial systems tested in literature at a fraction of the cost. The scores according to various classifi cation systems were determined and reported. Conclusion: Every platelet rich plasma prepared for therapeutic purposes should be classifi ed according to any of the available scoring systems to determine its suitability for the purpose and to maintain uniformity in the quality of the product. The proposed two step method of preparation yields satisfactory quality of PRP by utilizing services of a basic laboratory at a fraction of the cost of commercial systems thereby enabling smaller medical institutions to utilize PRP therapy.

The neuroprotective activity of heat-treated human platelet lysate biomaterials manufactured from outdated pathogen-reduced (amotosalen/UVA) platelet concentrates

Background Effective neurorestorative therapies of neurodegenerative diseases must be developed. There is increasing interest in using human platelet lysates, rich in neurotrophic factors, as novel disease-modifying strategy of neurodegeneration. To ensure virus safety, pathogen reduction treatments should be incorporated in the preparation process of the platelet concentrates used as source material. We therefore investigated whether platelet concentrates (PC) pathogen-inactivated using a licensed photo-inactivation treatment combining photosensitive psoralen (amotosalen) and UVA irradiation (Intercept) can serve as source material to prepare platelet lysates with preserved neuroprotective activity in Parkinson’s disease models. Methods Intercept treated-PCs were centrifuged, when reaching expiry day (7 days after collection), to remove plasma and platelet additive solution. The platelet pellet was re-suspended and concentrated in phosphate buffer saline, subjected to 3 freeze-thaw cycles (− 80 °C/37 °C) then centrifuged to remove cell debris. The supernatant was recovered and further purified, or not, by heat-treatment as in our previous investigations. The content in proteins and neurotrophic factors was determined and the toxicity and neuroprotective activity of the platelet lysates towards LUHMES cells or primary cortical/hippocampal neurons were assessed using ELISA, flow cytometry, cell viability and cytotoxicity assays and proteins analysis by Western blot. Results Platelet lysates contained the expected level of total proteins (ca. 7–14 mg/mL) and neurotrophic factors. Virally inactivated and heat-treated platelet lysates did not exert detectable toxic effects on neither Lund human mesencephalic dopaminergic LUHMES cell line nor primary neurons. When used at doses of 5 and 0.5%, they enhanced the expression of tyrosine hydroxylase and neuron-specific enolase in LUHMES cells and did not significantly impact synaptic protein expression in primary neurons, respectively. Furthermore, virally-inactivated platelet lysates tested were found to exert very strong neuroprotection effects on both LUHMES and primary neurons exposed to erastin, an inducer of ferroptosis cell death. Conclusion Outdated Intercept pathogen-reduced platelet concentrates can be used to prepare safe and highly neuroprotective human heat-treated platelet pellet lysates. These data open reassuring perspectives in the possibility to develop an effective biotherapy using virally-inactivated platelet lysates rich in functional neurotrophins for neuroregenerative medicine, and for further bio-industrial development. However, the data should be confirmed in animal models. Graphical abstract

A New Rapid Method to Measure Human Platelet Cholesterol

Introduction: Platelet cholesterol (PC) could be used to assess “tissue” cholesterol of patients with vascular disease. However, the methods available so far to measure PC involve a complex extraction process. We developed a rapid method to measure PC and assessed its correlation with serum total cholesterol (TC), low-density lipoprotein cholesterol (LDL-C), high-density lipoprotein cholesterol (HDL-C), LDL-C/HDL-C ratio, triglycerides (TG), and non-HDL-C. Methods: We assessed repeatability (20 times, 3 participants) and reproducibility (8 times, 2 participants). A group of 47 healthy participants was studied. Blood was collected to analyze serum TC, LDL-C, HDL-C, and TG. Citrated blood was used to prepare a platelet pellet. A “clear soup” was produced (by disrupting this pellet using freeze–thaw and sonication cycles) and used to measure PC. Results: Repeatability of PC showed a coefficient of variation (CV) of 4.8%. The reproducibility of PC over a period of 2 months was CV 7.5% and 8.1% (8 measurements for 2 participants). The PC of participants with serum LDL-C >2.6 mmol/L (treatment goal recommended by the National Cholesterol Education Program Adult Treatment Panel III) was 377 ± 120 μmol/10 12 platelets (n = 25). There was a significant correlation (Spearman, correlation coefficient) of PC (n = 25) with serum LDL-C ( rs = 0.45, P = .02), LDL-C/HDL-C ( rs = 0.45, P = .02), TG ( rs = 0.43, P = .03), and non-HDL-C ( rs = 0.53, P = .007). Conclusion: This technique of measuring PC has the advantage of being reproducible, fast, and simpler than previous methods. Thus, it may be useful for multiple sampling when investigating changes in PC in hypercholesterolemic patients. More extensive evaluation is necessary.

Thrombin-stimulated Human Platelets Express Molecular Forms of α-Granule Factor V (FV), which Differ from FVa

SummaryBinding of 125I-Fab fragments of chain-specific antibodies indicate that both heavy and light chains of a-granule factor Va (FVa) were externalized on the platelet membrane after stimulation with thrombin. Using a Mab against the activation peptide of factor V (FV), the epitope appears on the stimulated platelet surface. Half as much light chain and heavy chain (FVa) was expressed compared to the activation peptide, suggesting that expression of α-granule FV occurs after thrombin stimulation. Using an ELISA, we find that 32% of a-granule FV was released and 68% is retained in the platelet pellet. Immunoblots of platelets indicate that FV exists in 200 kDa und 150 kDa forms, representing incomplete cleavage, while the releasate demonstrates a more complete cleavage by proteases. We conclude that expression of α-granule FV is quantitatively greater than that released and exists in molecular forms which cannot be completely explained by the binding of FVa.