In-vitro production and pre-validation of lyophilized canine platelet-rich plasma for therapeutic use

2021 ◽  
Vol 41 ◽  
Author(s):  
Natália P.P. Freitas ◽  
Maria Márcia M.S. Maior ◽  
Beatriz A.P. Silva ◽  
Marcus R.L. Bezerra ◽  
José F. Nunes ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Cell Proliferation ◽  
Growth Factors ◽  
Platelet Rich Plasma ◽  
Three Dimensional ◽  
In Vitro Production ◽  
Mesh Structure ◽  
Prp Gel

ABSTRACT: Platelet-rich plasma (PRP) has been considered a promising therapeutic alternative, since platelets are rich in growth factors that are used in the Regenerative Medicine field. However, fresh PRP cannot be stored for long periods. This study aimed to develop a protocol for obtaining lyophilized canine PRP capable of maintaining viability after its reconstitution. For that purpose, canine PRP extraction and lyophilization protocols were initially tested. Subsequently, assays were carried out to quantify the growth factors VEGF and TGF-β, before and after the lyophilization process, gelation test and the three-dimensional gel structure analysis of the reconstituted lyophilized PRP by electron microscopy, as well as in vitro cell proliferation test in lyophilized PRP gel. Additionally, the immunogenicity test was performed, using allogeneic samples of lyophilized PRP. The results showed that the lyophilized PRP had adequate therapeutic concentrations of growth factors VEGF and TGF-β (9.1pg/mL and 6161.6pg/mL, respectively). The reconstituted PRP gel after lyophilization showed an in vitro durability of 10 days. Its electron microscopy structure was similar to that of fresh PRP. In the cell proliferation test, an intense division process was verified in mesenchymal stem cells (MSCs) through the three-dimensional mesh structure of the lyophilized PRP gel. The immunogenicity test showed no evidence of an immune reaction. The findings were promising, suggesting the possibility of having a lyophilized canine PRP that can be marketed. New in vivo and in vitro studies must be carried out for therapeutic confirmation.

scholarly journals Chondrocyte Spheroids Laden in GelMA/HAMA Hybrid Hydrogel for Tissue-Engineered Cartilage with Enhanced Proliferation, Better Phenotype Maintenance, and Natural Morphological Structure

Gels ◽  
2021 ◽  
Vol 7 (4) ◽  
pp. 247
Author(s):  
Guanhuier Wang ◽  
Yang An ◽  
Xinling Zhang ◽  
Pengbing Ding ◽  
Hongsen Bi ◽  
...  
Keyword(s):  
Tissue Engineering ◽  
Cell Proliferation ◽  
Cartilage Tissue Engineering ◽  
Three Dimensional ◽  
Morphological Structure ◽  
Research Direction ◽  
Cartilage Tissue ◽  
Hybrid Hydrogel

Three-dimensional cell-laden tissue engineering has become an extensive research direction. This study aimed to evaluate whether chondrocyte spheroids (chondro-spheroids) prepared using the hanging-drop method could develop better cell proliferation and morphology maintenance characteristics, and be optimized as a micro unit for cartilage tissue engineering. Chondro-spheroids were loaded into a cross-linkable hybrid hydrogel of gelatin methacrylate (GelMA) and hyaluronic acid methacrylate (HAMA) in vivo and in vitro. Cell proliferation, aggregation, cell morphology maintenance as well as cartilage-related gene expression and matrix secretion in vitro and in vivo were evaluated. The results indicated that compared with chondrocyte-laden hydrogel, chondro-spheroid-laden hydrogel enhanced proliferation, had better phenotype maintenance, and a more natural morphological structure, which made it appropriate for use as a micro unit in cartilage tissue engineering.

scholarly journals Characterization of Fibroblasts with Son of Sevenless-1 Mutation

2006 ◽  
Vol 85 (11) ◽  
pp. 1050-1055 ◽  
Author(s):  
E.J. Lee ◽  
S.I. Jang ◽  
D. Pallos ◽  
J. Kather ◽  
T.C. Hart
Keyword(s):  
Cell Proliferation ◽  
Three Dimensional ◽  
Collagen Matrix ◽  
Histological Assessment ◽  
M Phase ◽  
Son Of Sevenless ◽  
Gingival Fibromatosis

Although non-syndromic hereditary gingival fibromatosis (HGF) is genetically heterogeneous, etiologic mutations have been identified only in the Son of Sevenless-1 gene ( SOS1). To test evidence of increased cell proliferation, we studied histological, morphological, and proliferation characteristics in monolayer and three-dimensional cultures of fibroblasts with the SOS1 g.126,142–126,143insC mutation. Histological assessment of HGF gingiva indicated increased numbers of fibroblasts (30%) and increased collagen (10%). Cell proliferation studies demonstrated increased growth rates and 5-bromo-2-deoxyuridine incorporation for HGF fibroblasts. Flow cytometry showed greater proportions of HGF fibroblasts in the G2/M phase. Attachment of HGF fibroblasts to different extracellular matrix surfaces demonstrated increased formation of protrusions with lamellipodia. HGF fibroblasts in three-dimensional culture showed greater cell proliferation, higher cell density, and alteration of surrounding collagen matrix. These findings revealed that increased fibroblast numbers and collagen matrix changes are associated with mutation of the SOS1 gene in vitro and in vivo.

scholarly journals Platelet-Rich Plasma in Bone Regeneration: Engineering the Delivery for Improved Clinical Efficacy

2014 ◽  
Vol 2014 ◽  
pp. 1-15 ◽  
Author(s):  
Isaac A. Rodriguez ◽  
Emily A. Growney Kalaf ◽  
Gary L. Bowlin ◽  
Scott A. Sell
Keyword(s):  
Growth Factors ◽  
Growth Factor ◽  
Bone Regeneration ◽  
Critical Size ◽  
Platelet Rich Plasma ◽  
Clinical Use ◽  
Regenerative Capacity ◽  
Effective Delivery

Human bone is a tissue with a fairly remarkable inherent capacity for regeneration; however, this regenerative capacity has its limitations, and defects larger than a critical size lack the ability to spontaneously heal. As such, the development and clinical translation of effective bone regeneration modalities are paramount. One regenerative medicine approach that is beginning to gain momentum in the clinical setting is the use of platelet-rich plasma (PRP). PRP therapy is essentially a method for concentrating platelets and their intrinsic growth factors to stimulate and accelerate a healing response. While PRP has shown some efficacy in bothin vitroandin vivoscenarios, to date its use and delivery have not been optimized for bone regeneration. Issues remain with the effective delivery of the platelet-derived growth factors to a localized site of injury, the activation and temporal release of the growth factors, and the rate of growth factor clearance. This review will briefly describe the physiological principles behind PRP use and then discuss how engineering its method of delivery may ultimately impact its ability to successfully translate to widespread clinical use.

scholarly journals Interpenetrating Hydrogel Networks Enhance Mechanical Stability, Rheological Properties, Release Behavior and Adhesiveness of Platelet-Rich Plasma

2020 ◽  
Vol 21 (4) ◽  
pp. 1399 ◽  
Author(s):  
Roberta Censi ◽  
Cristina Casadidio ◽  
Siyuan Deng ◽  
Maria Rosa Gigliobianco ◽  
Maria Giovanna Sabbieti ◽  
...  
Keyword(s):  
Growth Factors ◽  
Mechanical Stability ◽  
Platelet Rich Plasma ◽  
Polymer Networks ◽  
Interpenetrating Polymer Networks ◽  
Cartilage Defects ◽  
Lytic Enzymes ◽  
Thermal Gelation

Platelet-rich plasma (PRP) has attracted much attention for the treatment of articular cartilage defects or wounds due to its intrinsic content of growth factors relevant for tissue repair. However, the short residence time of PRP in vivo, due to the action of lytic enzymes, its weak mechanical properties and the consequent short-term release of bioactive factors has restricted its application and efficacy. The present work aimed at designing new formulation strategies for PRP, based on the use of platelet concentrate (PC)-loaded hydrogels or interpenetrating polymer networks, directed at improving mechanical stability and sustaining the release of bioactive growth factors over a prolonged time-span. The interpenetrating hydrogels comprised two polymer networks interlaced on a molecular scale: (a) a first covalent network of thermosensitive and biodegradable vinyl sulfone bearing p(hydroxypropyl methacrylamide-lacate)-polyethylene glycol triblock copolymers, tandem cross-linked by thermal gelation and Michael addition when combined with thiolated hyaluronic acid, and (b) a second network composed of cross-linked fibrin. The PC-loaded hydrogels, instead, was formed only by network (a). All the designed and successfully synthesized formulations greatly increased the stability of PRP in vitro, leading to significant increase in degradation time and storage modulus of PRP gel. The resulting viscoelastic networks showed the ability to controllably release platelet derived growth factor and transforming growth factr β1, and to improve the tissue adhesiveness of PRP. The newly developed hydrogels show great potential for application in the field of wound healing, cartilage repair and beyond.

scholarly journals CBMT-46. MICRORNA-206 ATTENUATES GLIOMA CELL PROLIFERATION, MIGRATION, AND INVASION BY BLOCKING THE WNT/β-CATENIN PATHWAY VIA DIRECT TARGETING OF FRIZZLED 7

2019 ◽  
Vol 21 (Supplement_6) ◽  
pp. vi43-vi43
Author(s):  
Yingyi Wang ◽  
Fengqi Zhou ◽  
Chunsheng Zhao
Keyword(s):  
Cell Proliferation ◽  
Cellular Proliferation ◽  
Three Dimensional ◽  
Inhibitory Effect ◽  
Cell Counting ◽  
Migration And Invasion ◽  
Nude Mouse Model ◽  
Real Time Quantitative Pcr

Abstract Glioma is one of the most frequent primary malignant brain tumours in adults and accumulating evidence has shown that microRNAs (miRNAs) are associated with various types of tumours, including glioma. It is essential to acquire a better understanding of the roles and mechanisms of miRNAs in WNT-driven glioblastoma (GBM). Here, we report that miR-206 inhibited the WNT/β-catenin pathway by directly targeting Frizzled 7 (FZD7) and functioned as a tumour-suppressor in glioma. The expression of miR-206 in human samples and glioma cells was assessed by real-time quantitative PCR (RT-qPCR), fluorescence in situ hybridization (FISH), and histological analysis. Cell Counting Kit-8 (CCK-8), colony formation, EdU, flow cytometry, wound healing, transwell invasion, and three-dimensional migration were performed to observe cellular proliferation, migration, and invasion in vitro. The effects of miR-206 in vivo were investigated using a xenograft nude mouse model. miR-206 expression was significantly downregulated in glioma specimens. FZD7 was confirmed as a direct target of miR-206. GBM cell proliferation, migration, and invasion were blocked by restoring the expression of miR-206. Moreover, intracranial glioma models demonstrated an inhibitory effect of miR-206 on intracranial glioma tumour growth. Our results suggested that miR-206 plays a key role in blocking the WNT/β-catenin signalling pathway by suppressing FZD7 and provides a prospective therapeutic strategy for the treatment of malignant glioma and other WNT-driven tumours.

scholarly journals Controlled release of basic fibroblast growth factor from a peptide biomaterial for bone regeneration

2020 ◽  
Vol 7 (4) ◽  
pp. 191830 ◽  
Author(s):  
WeiKang Zhao ◽  
Yuling Li ◽  
Ao Zhou ◽  
Xiaojun Chen ◽  
Kai Li ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Bone Repair ◽  
Three Dimensional ◽  
Controlled Drug Release ◽  
Confocal Laser ◽  
Polyamide 66 ◽  
Bone Mesenchymal Stem Cells ◽  
Alizarin Red

Self-assembled peptide scaffolds based on D-RADA16 are an important matrix for controlled drug release and three-dimensional cell culture. In this work, D-RADA16 peptide hydrogels were coated on artificial bone composed of nano-hydroxyapatite/polyamide 66 (nHA/PA66) to obtain a porous drug-releasing structure for treating bone defects. The developed materials were characterized via transmission electron microscopy and scanning electron microscopy. The proliferation and adhesion of bone mesenchymal stem cells (BMSCs) were examined by confocal laser microscopy and CCK-8 experiments. The osteogenic ability of the porous materials towards bone BMSCs was examined in vitro by staining with Alizarin Red S and alkaline phosphatase, and bioactivity was evaluated in vivo . The results revealed that nHA/PA66/D-RADA16/bFGF reduces the degradation rate of D-RADA16 hydrogels and prolongs sustained release of bFGF, which would promote BMSCs proliferation, adhesion and osteogenesis in vitro and bone repair in vivo . Thus, it deserves more attention and is worthy of further research.

The degree of inhibition of thyroid follicular cell proliferation by iodide is a highly individual characteristic of each cell and differs profoundly in vitro and in vivo

1994 ◽  
Vol 130 (6) ◽  
pp. 595-600 ◽  
Author(s):  
Simon Aeschimann ◽  
Hans Gerber ◽  
Christine von Grünigen ◽  
Madeleine Oestreicher ◽  
Hugo Studer
Keyword(s):  
Cell Proliferation ◽  
Three Dimensional ◽  
Inhibitory Effect ◽  
Individual Characteristic ◽  
Follicular Cell ◽  
Thyroid Follicular Cell ◽  
Inhibiting Effect ◽  
Growth Inhibiting

Aeschimann S, Gerber H, von Grünigen C, Oestreicher M, Studer H. The degree of inhibition of thyroid follicular cell proliferation by iodide is a highly individual characteristic of each cell and differs profoundly in vitro and in vivo. Eur J Endocrinol 1944;130:595–600. ISSN 0804–4643 Pharmacological concentrations of iodide (>1 × 10−6 mol/l) are known to inhibit thyroid follicular cell growth in vitro. However, the inhibitory effect varies widely, depending on experimental conditions, and usually does not exceed 50%. We demonstrate that iodide (10−4 mol/l) inhibits the growth of FRTL-5 cells in different passages by 11–67%. When five subclones of FRTL-5 cells were compared to the wild type, iodide-induced growth inhibition varied between 25% and 46%. The individual degree of inhibition of each clone was reproducible in two subsequent passages, suggesting that it is a stable constitutive trait. When FRTL-5 cells were grown first in three-dimensional clusters and then transplanted onto nude mice with high endogenous thyrotropin secretion, iodide at a serum concentration of less than 5.7 × 10−7 mol/l nearly completely blocked cell replication in the transplants but not in the mice's own thyroid. Five cell lines, prepared from autonomously growing hyperthyroid feline multinodular goiters, were nearly completely resistant to the growth-inhibitory effect of iodide. These observations suggest that the sensitivity towards the growth-inhibiting effect of iodide is a highly variable, stable trait of each thyrocyte, even in cloned cell populations. Some FRTL-5 cells and, even more so, cells prepared from autonomously growing nodular feline goiters are resistant constitutively to the growth-inhibiting effect of iodide. Three-dimensional clusters of FRTL-5 cells growing as transplants in recipient mice are growth-arrested totally by iodide concentrations more than 175 times lower than those partially inhibiting monolayer cultures, while growth of the intact thyroid of the recipient mouse remains unaffected or is even enhanced. Thus, the sensitivity of thyrocytes toward iodide is different fundamentally in vitro and in vivo. Hugo Studer, Medizinische Universitaetsklinik, Inselspital, CH-3010 Bern, Switzerland

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