scholarly journals Novel leukocyte-depleted platelet-rich plasma-based skin equivalent as an in vitro model of chronic wounds: a preliminary study

2021 ◽  
Vol 22 (1) ◽  
Author(s):  
Elisa Seria ◽  
George Galea ◽  
Joseph Borg ◽  
Kevin Schembri ◽  
Gabriella Grech ◽  
...  
Keyword(s):  
Gene Expression ◽  
Wound Closure ◽  
Platelet Rich Plasma ◽  
Skin Equivalent ◽  
Skin Wounds ◽  
Skin Model ◽  
Scratch Assay ◽  
Skin Perfusion ◽  
Chronic Skin

Abstract Background Chronic leg ulcerations are associated with Haemoglobin disorders, Type2 Diabetes Mellitus, and long-term venous insufficiency, where poor perfusion and altered metabolism develop into a chronic inflammation that impairs wound closure. Skin equivalent organotypic cultures can be engineered in vitro to study skin biology and wound closure by modelling the specific cellular components of the skin. This study aimed to develop a novel bioactive platelet-rich plasma (PRP) leukocyte depleted scaffold to facilitate the study of common clinical skin wounds in patients with poor chronic skin perfusion and low leukocyte infiltration. A scratch assay was performed on the skin model to mimic two skin wound conditions, an untreated condition and a condition treated with recombinant tumour necrotic factor (rTNF) to imitate the stimulation of an inflammatory state. Gene expression of IL8 and TGFA was analysed in both conditions. Statistical analysis was done through ANOVA and paired student t-test. P < 0.05 was considered significant. Results A skin model that consisted of a leukocyte-depleted, platelet-rich plasma scaffold was setup with embedded fibroblasts as dermal equivalents and seeded keratinocytes as multi-layered epidermis. Gene expression levels of IL8 and TGFA were significantly different between the control and scratched conditions (p < 0.001 and p < 0.01 respectively), as well as between the control and treated conditions (p < 0.01 and p < 0.001 respectively). The scratch assay induced IL8 upregulation after 3 h (p < 0.05) which continued to increase up to day 1 (p < 0.05). On the other hand, the administration of TNF led to the downregulation of IL8 (p < 0.01), followed by an upregulation on day 2. IL8 gene expression decreased in the scratched condition after day 1 as the natural healing process took place and was lower than in the treated condition on day 8 (p < 0.05). Both untreated and treated conditions showed a downregulation of TGFA 3 h after scratch when compared with the control condition (p < 0.01). Administration of rTNF showed significant downregulation of TGFA after 24 h when compared with the control (p < 0.01) and treated conditions (p < 0.05). Conclusion This study suggests that a leukocyte-depleted PRP-based skin equivalent can be a useful model for the in vitro study of chronic skin wounds related to poor skin perfusion.

scholarly journals Mesenchymal Stem Cells Regulate IL8 and TGFA Expression in a Novel Leucocytes Depleted Platelet-Rich Plasma-Skin Equivalent in a Preliminary in vitro Study of Chronic Wound Healing

2020 ◽  
Author(s):  
Elisa Seria ◽  
George Galea ◽  
Gabriella Grech ◽  
Sarah Samut Tagliaferro ◽  
Alexander Felice
Keyword(s):  
Stem Cells ◽  
Mesenchymal Stem Cells ◽  
Chronic Wounds ◽  
Platelet Rich Plasma ◽  
Healing Process ◽  
In Vitro Study ◽  
Skin Equivalent ◽  
Skin Perfusion ◽  
Inflammatory State

Abstract Background: Chronic leg ulcerations are associated with Haemoglobin disorders, Type 2 Diabetes Mellitus, and long-term venous insufficiency. Mesenchymal stem cells (MSCs) ability to modulate the inflammatory response represents the fundamental requisite for their applicability as a treatment of chronic wounds.Methods: This study aimed to develop a novel bioactive platelet-rich plasma (PRP)-leukocytes-depleted scaffold to reproduce typical clinical wound of patients with poor chronic skin perfusion and low leucocytes infiltration. After scratching the wound model to mimic injury three conditions were compared; an untreated condition, a condition treated with recombinant TNF to mimic an inflammatory state and a condition treated with TNF and also with MSCs to evaluate how the latter’s immunomodulatory properties affect the therapeutic outcomes in an inflammatory state. Gene expression of IL8 and TGFA were analysed in biological triplicates of the three conditions. Statistical analysis was done through a paired student t-test and a p <0.05 was considered significant.Results: We set up a skin model that consisted of a leukocyte-depleted, platelet-rich plasma scaffold, with embedded fibroblasts as dermal equivalent and seeded keratinocytes on it as multi-layered epidermidis. IL8 expression increased upon scratching (p=0.014) and continued to increase up to day 1 (p=0.048). IL8 expression decreased upon administration of TNF (p=0.005) but then increased again. IL8 expression decreased in the untreated condition after day 1 as the natural healing process took place and was lower than in treated conditions in day 8 (p=0.048). TGFA expression decreased upon scratching (p=0.006) and increased again in day 1, more so in the untreated than in the treated conditions (p=0.02). TGFA expression decreased again in day 4 in the study group before increasing sharply (p=0.027) in day 8 to reach pre-scratch levels. Conclusion: This study found that a leukocyte-depleted PRP-based skin equivalent can be useful in the study of treatments of chronic wounds. This study also indicates that MSCs appear to modulate the expression of IL8 by switching from an immunosuppressive phenotype to a pro-inflammatory phenotype. These results indicate that the administration of MSCs could offer a potential therapeutic approach for the treatment of leg ulcers in patients with poor skin perfusion.

Anti-Inflammatory Effects of Novel Standardized Platelet Rich Plasma Releasates on Knee Osteoarthritic Chondrocytes and Cartilage in vitro

2019 ◽  
Vol 20 (11) ◽  
pp. 920-933 ◽  
Author(s):  
Lucía Gato-Calvo ◽  
Tamara Hermida-Gómez ◽  
Cristina R. Romero ◽  
Elena F. Burguera ◽  
Francisco J. Blanco
Keyword(s):  
Gene Expression ◽  
Ex Vivo ◽  
Platelet Rich Plasma ◽  
Cartilage Explants ◽  
Ex Vivo Model ◽  
Chondrocyte Viability ◽  
Platelet Concentration ◽  
The Absolute ◽  
Anti Inflammatory

Background: Platelet Rich Plasma (PRP) has recently emerged as a potential treatment for osteoarthritis (OA), but composition heterogeneity hampers comparison among studies, with the result that definite conclusions on its efficacy have not been reached. Objective: 1) To develop a novel methodology to prepare a series of standardized PRP releasates (PRP-Rs) with known absolute platelet concentrations, and 2) To evaluate the influence of this standardization parameter on the anti-inflammatory properties of these PRP-Rs in an in vitro and an ex vivo model of OA. Methods: A series of PRPs was prepared using the absolute platelet concentration as the standardization parameter. Doses of platelets ranged from 0% (platelet poor plasma, PPP) to 1.5·105 platelets/µl. PRPs were then activated with CaCl2 to obtain releasates (PRP-R). Chondrocytes were stimulated with 10% of each PRP-R in serum-free culture medium for 72 h to assess proliferation and viability. Cells were co-stimulated with interleukin (IL)-1β (5 ng/ml) and 10% of each PRP-R for 48 h to determine the effects on gene expression, secretion and intra-cellular content of common markers associated with inflammation, catabolism and oxidative stress in OA. OA cartilage explants were co-stimulated with IL-1β (5 ng/ml) and 10% of either PRP-R with 0.75·105 platelets/µl or PRP-R with 1.5·105 platelets/µl for 21 days to assess matrix inflammatory degradation. Results: Chondrocyte viability was not affected, and proliferation was dose-dependently increased. The gene expression of all pro-inflammatory mediators was significantly and dose-independently reduced, except for that of IL-1β and IL-8. Immunoblotting corroborated this effect for inducible NO synthase (NOS2). Secreted matrix metalloproteinase-13 (MMP-13) was reduced to almost basal levels by the PRP-R from PPP. Increasing platelet dosage led to progressive loss to this anti-catabolic ability. Safranin O and toluidine blue stains supported the beneficial effect of low platelet dosage on cartilage matrix preservation. Conclusion: We have developed a methodology to prepare PRP releasates using the absolute platelet concentration as the standardization parameter. Using this approach, the composition of the resulting PRP derived product is independent of the donor initial basal platelet count, thereby allowing the evaluation of its effects objectively and reproducibly. In our OA models, PRP-Rs showed antiinflammatory, anti-oxidant and anti-catabolic properties. Platelet enrichment could favor chondrocyte proliferation but is not necessary for the above effects and could even be counter-productive.

scholarly journals Systematic Review: Adipose-Derived Mesenchymal Stem Cells, Platelet-Rich Plasma and Biomaterials as New Regenerative Strategies in Chronic Skin Wounds and Soft Tissue Defects

2021 ◽  
Vol 22 (4) ◽  
pp. 1538 ◽  
Author(s):  
Pietro Gentile ◽  
Simone Garcovich
Keyword(s):  
Systematic Review ◽  
Stem Cells ◽  
Wound Healing ◽  
Soft Tissue ◽  
Chronic Wounds ◽  
Platelet Rich Plasma ◽  
Skin Wounds ◽  
Soft Tissue Defects ◽  
Chronic Skin ◽  
Tissue Defects

The number of clinical trials evaluating adipose-derived mesenchymal stem cells (AD-MSCs), platelet-rich plasma (PRP), and biomaterials efficacy in regenerative plastic surgery has exponentially increased during the last ten years. AD-MSCs are easily accessible from various fat depots and show intrinsic plasticity in giving rise to cell types involved in wound healing and angiogenesis. AD-MSCs have been used in the treatment of soft tissue defects and chronic wounds, employed in conjunction with a fat grafting technique or with dermal substitute scaffolds and platelet-rich plasma. In this systematic review, an overview of the current knowledge on this topic has been provided, based on existing studies and the authors’ experience. A multistep search of the PubMed, MEDLINE, Embase, PreMEDLINE, Ebase, CINAHL, PsycINFO, Clinicaltrials.gov, Scopus database, and Cochrane databases has been performed to identify papers on AD-MSCs, PRP, and biomaterials used in soft tissue defects and chronic wounds. Of the 2136 articles initially identified, 422 articles focusing on regenerative strategies in wound healing were selected and, consequently, only 278 articles apparently related to AD-MSC, PRP, and biomaterials were initially assessed for eligibility. Of these, 85 articles were excluded as pre-clinical, experimental, and in vitro studies. For the above-mentioned reasons, 193 articles were selected; of this amount, 121 letters, expert opinions, commentary, and editorials were removed. The remaining 72 articles, strictly regarding the use of AD-MSCs, PRP, and biomaterials in chronic skin wounds and soft tissue defects, were analyzed. The studies included had to match predetermined criteria according to the patients, intervention, comparator, outcomes, and study design (PICOS) approach. The information analyzed highlights the safety and efficacy of AD-MSCs, PRP, and biomaterials on soft tissue defects and chronic wounds, without major side effects.

scholarly journals Optimization of Lyophilized Hyperacute Serum (HAS) as a Regenerative Therapeutic in Osteoarthritis

2021 ◽  
Vol 22 (14) ◽  
pp. 7496
Author(s):  
Isabel Olmos Calvo ◽  
Olga Kuten-Pella ◽  
Karina Kramer ◽  
Ágnes Madár ◽  
Szilvia Takács ◽  
...  
Keyword(s):  
Gene Expression ◽  
Gene Expression Analysis ◽  
Platelet Rich Plasma ◽  
Cell Types ◽  
Inflammatory Conditions ◽  
Osteoarthritic Chondrocytes ◽  
Prp Gene ◽  
Human Explants ◽  
Cytokine Quantification

Hyperacute serum (HAS) is a blood derivative product that promotes the proliferation of various cell types and controls inflammation in vitro. The aim of this study is to investigate the regenerative potential of different formulations of HAS, including lyophilized and hyaluronic acid combined versions, to obtain a stable and standardized therapeutic in osteoarthritis (OA), which may be able to overcome the variability limitations of platelet-rich plasma (PRP). Primary human osteoarthritic chondrocytes were used for testing cellular viability and gene expression of OA-related genes. Moreover, a co-culture of human explants of cartilage, bone and synovium under inflammatory conditions was used for investigating the inflammatory control capacities of the different therapeutics. In this study, one formulation of lyophilized HAS achieved the high cell viability rates of liquid HAS and PRP. Gene expression analysis showed that HAS induced higher Col1a1 expression than PRP. Cytokine quantification from supernatant fluids revealed that HAS treatment of inflamed co-cultures significantly reduced levels of IL-5, IL-15, IL-2, TNFα, IL-7 and IL-12. To conclude, lyophilized HAS is a stable and standardized therapeutic with high potential in joint regeneration.

scholarly journals Effects of platelet-rich plasma on in vitro proliferation and migration of fibroblasts from human chronic refractory wound granulation tissue

2021 ◽  
Vol 8 (1) ◽  
pp. 24
Author(s):  
Tie-ning Zhang ◽  
Quan Li ◽  
Te Ba ◽  
Tian-xi Shao ◽  
Fang Li ◽  
...  
Keyword(s):  
Granulation Tissue ◽  
Platelet Rich Plasma ◽  
Human Fibroblasts ◽  
In Vitro Proliferation ◽  
Scratch Assay ◽  
Fetal Bovine ◽  
And Migration ◽  
Wound Scratch Assay ◽  
Proliferation And Migration

Objective: To observe the effects of platelet-rich plasma (PRP) on in vitro proliferation and migration of fibroblasts from human chronic refractory wound granulation tissue.Methods: Fibroblasts were separated from human chronic refractory wound granulation tissue and then were identified. The obtained fibroblasts were divided into fetal bovine serum (FBS) group, hydrogel group and PRP group, and the three groups were cultured with culture mediums containing FBS, hydrogel and PRP respectively, in order to observe the growth of fibroblasts. The wound scratch assay was used to observe the migration of fibroblasts.Results: PRP group had more fibroblasts than FBS group and hydrogel group since Day 5 of culture, and exhibited greater fibroblast scratch migration area than FBS group on 48 h and 72 h of wound scratch assay (all p < .05).Conclusions: Compared with FBS, human fibroblasts cultured by PRP can more effectively promote the proliferation and migration of fibroblasts.

scholarly journals The opioid peptide biphalin modulates human corneal epithelial wound healing in vitro

2019 ◽  
Author(s):  
Ozgun Melike Gedar Totuk ◽  
Erdost YILDIZ ◽  
Adriano MOLLICA ◽  
Kerem KABADAYI ◽  
Afsun SAHIN
Keyword(s):  
Wound Healing ◽  
Corneal Epithelium ◽  
Wound Closure ◽  
Opioid Peptide ◽  
Corneal Epithelial Cell ◽  
Scratch Assay ◽  
Corneal Epithelial ◽  
Significant Difference ◽  
Epithelial Wound Healing

Abstract Background Analgesic drugs including nonselective opioids and non-steroidal anti-inflammatory drugs should be used with great precautions to relieve the pain after physical damage of the corneal epithelium, because of their unfavorable effects on wound healing process. Biphalin, a synthetic opioid peptide, which has been demonstrated to possesses a strong analgesic effect on rodents. The purpose of this study is to investigate the effects of biphalin on human corneal epithelium wound healing. Methods Immortalized human corneal epithelial cell (HCEC) culture was used to test the effects of biphalin on wound healing. The toxicity of biphalin in various concentrations was measured with MTT assay. The effect of 1 µM and 10 µM biphalin were tested on wound closure at in vitro scratch assay of HCECs, and for cell migration and proliferation separately. Naloxone, a non-selective competitive antagonist of opioid receptor, was also used to inhibit the effects of biphalin in all experiments. Results Biphalin did not cause any toxic effect on HCECs in concentrations lower than 100 µM at various incubation time points. Biphalin increased wound closure process significantly at 1 µM concentration at in vitro scratch assay of HCECs (p < 0.05); also increased migration of HCECs significantly (p < 0.01). There was no significant difference between biphalin and control groups of HCECs at Ki67 proliferation assay. Conclusion Biphalin, a synthetic opioid peptide, has a potential role as a novel topical analgesic agent that promotes corneal epithelial wound healing.

Conditioned Medium of Human Adipose Mesenchymal Stem Cells Increases Wound Closure and Protects Human Astrocytes Following Scratch Assay In Vitro

2017 ◽  
Vol 55 (6) ◽  
pp. 5377-5392 ◽  
Author(s):  
Eliana Baez-Jurado ◽  
Oscar Hidalgo-Lanussa ◽  
Gina Guio-Vega ◽  
Ghulam Md Ashraf ◽  
Valentina Echeverria ◽  
...  
Keyword(s):  
Stem Cells ◽  
Mesenchymal Stem Cells ◽  
Conditioned Medium ◽  
Wound Closure ◽  
Scratch Assay ◽  
Human Astrocytes ◽  
Adipose Mesenchymal Stem Cells

Selection of Keratinocytes Transduced with the Multidrug Resistance Gene in an in Vitro Skin Model Presents a Strategy for Enhancing Gene Expression in Vivo

1999 ◽  
Vol 10 (17) ◽  
pp. 2811-2821 ◽  
Author(s):  
Wolfgang Pfutzner ◽  
Ulrich R. Hengge ◽  
Mohamed A. Joari ◽  
Ruth-Ann Foster ◽  
Jonathan C. Vogel
Keyword(s):  
Gene Expression ◽  
Multidrug Resistance ◽  
Resistance Gene ◽  
Skin Model ◽  
Multidrug Resistance Gene ◽  
Selection Of ◽  
In Vitro Skin Model

scholarly journals Platelet-Released Growth Factors Modulate the Secretion of Cytokines in Synoviocytes under Inflammatory Joint Disease

2017 ◽  
Vol 2017 ◽  
pp. 1-9 ◽  
Author(s):  
Mersedeh Tohidnezhad ◽  
Andreas Bayer ◽  
Biljana Rasuo ◽  
Jennifer Vanessa Phi Hock ◽  
Nisreen Kweider ◽  
...  
Keyword(s):  
Gene Expression ◽  
Platelet Rich Plasma ◽  
Joint Disease ◽  
Cytokine Gene Expression ◽  
Inflammatory Conditions ◽  
Autologous Platelet Rich Plasma ◽  
Exact Effect ◽  
Autologous Platelet ◽  
Inflammatory Effect

The etiology and pathogenesis of rheumatoid arthritis (RA) are marked by a complex interplay of various cell populations and is mediated by different signaling pathways. Traditionally, therapies have primarily focused on pain relief, reducing inflammation and the recovery of joint function. More recently, however, researchers have discussed the therapeutic efficacy of autologous platelet-rich plasma (PRP). The main objective of this work is to examine the influences of platelet-released growth factor (PRGF) on human synoviocytes under inflammatory conditions. Additionally, it is checked to which extend treatment with platelet concentrate influences the release of cytokines form synoviocytes. For this purpose, an in vitro RA model was created by stimulating the cells with the TNF-α. The release of cytokines was measured by ELISA. The cytokine gene expression was analyzed by real-time PCR. It has been observed that the stimulation concentration of 10 ng/ml TNF-α resulted in a significantly increased endogenous secretion and gene expression of IL-6 and TNF-α. The anti-inflammatory effect of PRGF could be confirmed through significant reduction of TNF-α and IL-1β. An induced inflammatory condition seems to cause PRGF to inhibit the release of proinflammatory cytokines. Further study is required to understand the exact effect mechanism of PRGF on synoviocytes.

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