[Show abstract][Hide abstract] ABSTRACT: To evaluate the efficacy of platelet-rich plasma regarding healing, pain and hemostasis after total knee arthroplasty, by means of a blinded randomized controlled and blinded clinical study.
Forty patients who were going to undergo implantation of a total knee prosthesis were selected and randomized. In 20 of these patients, platelet-rich plasma was applied before the joint capsule was closed. The hemoglobin (mg/dL) and hematocrit (%) levels were assayed before the operation and 24 and 48 h afterwards. The Womac questionnaire and a verbal pain scale were applied and knee range of motion measurements were made up to the second postoperative month. The statistical analysis compared the results with the aim of determining whether there were any differences between the groups at each of the evaluation times.
The hemoglobin (mg/dL) and hematocrit (%) measurements made before the operation and 24 and 48 h afterwards did not show any significant differences between the groups (p > 0.05). The Womac questionnaire and the range of motion measured before the operation and up to the first two months also did not show any statistical differences between the groups (p > 0.05). The pain evaluation using the verbal scale showed that there was an advantage for the group that received platelet-rich plasma, 24 h, 48 h, one week, three weeks and two months after the operation (p < 0.05).
In the manner in which the platelet-rich plasma was used, it was not shown to be effective for reducing bleeding or improving knee function after arthroplasty, in comparison with the controls. There was an advantage on the postoperative verbal pain scale.
[Show abstract][Hide abstract] ABSTRACT: The use of mesenchymal stem cells (MSCs) in experimental, clinical, and therapeutic trials has grown in recent years. However, the issue remains of whether these procedures are completely safe for transplant patients. Therefore, this study was designed and carried out with the aim of evaluating two different comet assay protocols for genomic damage pattern analysis in MSCs derived from adipose tissue. The analyzed and interpreted results suggest that genetic testing is needed to support clonal expansion safety in cell therapy procedures with MSCs. Furthermore, they also suggest that if the comet assay technique would be used as a genomic integrity screening assay, the protocol performed at pH = 12 (that yielded a frequency of damaged cells: tail intensity = 9.50 ± 0.60, tail moment = 0.0122 ± 0.0007; results are reported as means ± standard deviation) would be indicated as genomic damage, and that subsequent single-strand breaks occur at pH > 13 (frequency of damaged cells: tail intensity = 30.71 ± 4.23, tail moment = 0.0447 ± 0.0073). Our study demonstrates that, in the era of regenerative medicine, it is necessary to standardize and establish a battery of tests in order to identify genomic damage prior to MSC transplantation.
Full-text · Article · Jan 2015 · Genetics and molecular research: GMR
[Show abstract][Hide abstract] ABSTRACT: Treatments for injured articular cartilage have not advanced to the point that efficient regeneration is possible. However, there has been an increase in the use of platelet-rich plasma for the treatment of several orthopedic disorders, including chondral injuries. Our hypothesis is that the treatment of chondral injuries with platelet gel results in higher-quality repair tissue after 180 days compared with chondral injuries not treated with gel.
A controlled experimental laboratory study was performed on 30 male rabbits to evaluate osteochondral injury repair after treatment with or without platelet gel. Osteochondral injuries were surgically induced in both knees of each rabbit at the medial femoral condyle. The left knee injury was filled with the platelet gel, and the right knee was not treated. Microscopic analysis of both knee samples was performed after 180 days using a histological grading scale.
The only histological evaluation criterion that was not significantly different between treatments was metachromasia. The group that was treated with platelet gel exhibited superior results in all other criteria (cell morphology, surface regularity, chondral thickness and repair tissue integration) and in the total score.
The repair tissue was histologically superior after 180 days in the study group treated with platelet gel compared with the group of untreated injuries.
Preview · Article · Dec 2014 · Clinics (São Paulo, Brazil)
[Show abstract][Hide abstract] ABSTRACT: Cytokines are small glycoproteins, weighting from 8 to 30 kDa, important in cell signaling. Not as classical hormones, cytokines are not stored as preformed molecules, acting especially by paracrine (in neighboring cells) and autocrine (in the producing cells) mechanisms. They influence the activity, differentiation, proliferation, and survival of immune cells, as well as regulate the production and activity of other cytokines that can increase or decrease the inflammatory response. Some cytokines can have a pro (Th1) or anti-inflammatory (Th2) actions, according to the microenvironment in which they are located. Among proinflammatory cytokines, we can mention interleukins (IL)1,2,6,7 and TNF (tumor necrosis factor). Anti-inflammatory cytokines include IL-4,IL-10,IL-13,and TGFβ (transforming growth factor). Scaffolds are three dimensional (3D) structures developed to promote mechanical support and favorable microenvironment to cells, this technology opens new gates to regenerative medicine. Adipose tissue represents an accessible and promising source of adult mesenchymal stem cells (ADSCs) providing good number and concentration. This and lymphocytes. The aim of this study was to analyze the interaction between adipose derived stem cells, fibroblasts and three types of bioscaffolds developed in Botucatu's School of Medicine Blood Center composed by fibrin glue, platelet gel and chitosan incorporated with platelet derived hormones. The study was designed to access and measure some cytokines (interleukin released on the medium supernatant of cells in contact with scaffolds). Two samples of ADSCs and fibroblasts were cultivated into this scaffolds and the cytokines interleukin IL-8, IL-1β, IL-6, IL-10, tumorigenesis/necrosis(TNF), and IL-12p70 were accessed by Human Inflammatory Cytokines kit (CBA) -BD ® ,analysis using FCAPArray software BD ® by flow cytometry method, by harvesting supernatant.
[Show abstract][Hide abstract] ABSTRACT: Objective
To evaluate the efficacy of platelet‐rich plasma regarding healing, pain and hemostasis after total knee arthroplasty, by means of a blinded randomized controlled and blinded clinical study.
Forty patients who were going to undergo implantation of a total knee prosthesis were selected and randomized. In 20 of these patients, platelet‐rich plasma was applied before the joint capsule was closed. The hemoglobin (mg/dL) and hematocrit (%) levels were assayed before the operation and 24 and 48 hours afterwards. The Womac questionnaire and a verbal pain scale were applied and knee range of motion measurements were made up to the second postoperative month. The statistical analysis compared the results with the aim of determining whether there were any differences between the groups at each of the evaluation times.
The hemoglobin (mg/dL) and hematocrit (%) measurements made before the operation and 24 and 48 hours afterwards did not show any significant differences between the groups (p > 0.05). The Womac questionnaire and the range of motion measured before the operation and up to the first two months also did not show any statistical differences between the groups (p > 0.05). The pain evaluation using the verbal scale showed that there was an advantage for the group that received platelet‐rich plasma, 24 hours, 48 hours, one week, three weeks and two months after the operation (p < 0.05).
In the manner in which the platelet‐rich plasma was used, it was not shown to be effective for reducing bleeding or improving knee function after arthroplasty, in comparison with the controls. There was an advantage on the postoperative verbal pain scale.
Preview · Article · Sep 2014 · Revista Brasileira de Ortopedia
[Show abstract][Hide abstract] ABSTRACT: Purpose:
To obtain a decellularized tracheal scaffold associating traditional approaches with the novel light-emitting diode (LED) proposal.
This study was performed with New Zealand adult rabbits weighing 3.0 - 4.0 kg. Different protocols (22) were used combining physical (agitation and LED irradiation), chemical (SDS and Triton X-100 detergents), and enzymatic methods (DNase and RNase).
Generally, the cells surrounding soft tissues were successfully removed, but none protocol removed cells from the tracheal cartilage. However, longer protocols were more effective. The cost-benefits relation of the enzymatic processes was not favorable. It was possible to find out that the cartilaginous tissue submitted to the irradiation with LED 630nm and 475 nm showed an increased number of gaps without cells, but several cells were observed to be still present.
The light-emitting diode is a promising tool for decellularization of soft tissues.
Full-text · Article · Aug 2014 · Acta cirurgica brasileira / Sociedade Brasileira para Desenvolvimento Pesquisa em Cirurgia
[Show abstract][Hide abstract] ABSTRACT: OBJECTIVES:
Despite the recent success regarding the transplantation of tissue-engineered airways, the mechanical properties of these grafts are not well understood. Mechanical assessment of a tissue-engineered airway graft before implantation may be used in the future as a predictor of function. The aim of this preliminary work was to develop a noninvasive image-processing environment for the assessment of airway mechanics.
Decellularized, recellularized and normal tracheas (groups DECEL, RECEL, and CONTROL, respectively) immersed in Krebs-Henseleit solution were ventilated by a small-animal ventilator connected to a Fleisch pneumotachograph and two pressure transducers (differential and gauge). A camera connected to a stereomicroscope captured images of the pulsation of the trachea before instillation of saline solution and after instillation of Krebs-Henseleit solution, followed by instillation with Krebs-Henseleit with methacholine 0.1 M (protocols A, K and KMCh, respectively). The data were post-processed with computer software and statistical comparisons between groups and protocols were performed.
There were statistically significant variations in the image measurements of the medial region of the trachea between the groups (two-way analysis of variance [ANOVA], p<0.01) and of the proximal region between the groups and protocols (two-way ANOVA, p<0.01).
The technique developed in this study is an innovative method for performing a mechanical assessment of engineered tracheal grafts that will enable evaluation of the viscoelastic properties of neo-tracheas prior to transplantation.
[Show abstract][Hide abstract] ABSTRACT: Clinical experience for peripheral arterial disease treatment shows poor results when synthetic grafts are used to approach infrapopliteal arterial segments. However, tissue engeneering may be an option to yield surrogate biocompatible neovessels. Thus, biological decellularized scaffolds could provide natural tissue architecture to use in tissue engineering, when the absence of ideal autologous veins reduces surgical options. The goal of this study was to evaluate different chemical induced decellularization protocols of the inferior vena cava of rabbits. They were decellularized with Triton X100 (TX100), Sodium Dodecyl Sulphate (SDS) or Sodium Deoxycholate (DS). Afterwards, we assessed the remaining extracellular matrix (ECM) integrity, residual toxicity and the biomechanical resistance of the scaffolds. Our results showed that TX100 was not effective to remove the cells, while protocols using SDS 1% for 2h and DS 2% for 1h, efficiently removed the cells and were better characterized. These scaffolds preserved the original organization of ECM. Additionally, the residual toxicity assessment did not reveal statistically significant changes while decellularized scaffolds retained the equivalent biomechanical properties when compared with the control. Our results concluded that protocols using SDS and DS were effective at obtaining decellularized scaffolds, which may be useful for blood vessel tissue engineering.
Full-text · Article · Jun 2014 · Experimental Cell Research
[Show abstract][Hide abstract] ABSTRACT: Melatonin is a free radical scavenger with important actions in the study of renal ischemia and reperfusion (I/R). This study evaluated possible renal protection of high doses of melatonin in an experimental model of I/R in which rats were submitted to acute hyperglycemia under anesthesia with isoflurane.
No preview · Article · May 2014 · Transplantation Proceedings
[Show abstract][Hide abstract] ABSTRACT: The aim of this study was to evaluate, in vitro, the role of bFGF in the proliferation and expression of collagen type I and fibronectin of dog bone marrow mesenchymal stem cells (dBMMSCs) in comparison with the expression of the same proteins in dog periodontal fibroblasts (dPLFs).
dBMMSCs from the iliac crest were cultivated in Dulbecco's Modified Eagle's Medium (DMEM). Flow cytometry analysis (FCA) was used to characterize dBMMSC. Cells were stimulated with bFGF (1, 5 and 10ng/mL) after 24 and 48h. Real time RT-PCR was performed to verify collagen type I and fibronectin expressions. MTT assay was used to confirm cellular proliferation. Statistical analyses were performed (ANOVA and Kruskal-Wallis tests; p<0.05).
FCA showed 55.98% of CD34+ and 32.67% of CD90+ after bone marrow aspiration; 3.33% of CD34+ and 33.0% of CD90+ before P1. After P2, 10.54% of dBMMSCs expressed CD90, whereas after P3, this number decreased to 1.58%. dPLFs presented 4.04% of CD90+ and 1.05% of CD34+ after P3. MTT evaluation showed increase in dBMSC proliferation with 5ng/mL bFGF-stimulus after 24-h. Both collagen I and fibronectin expression were very similar between the two cells groups after 24-h stimulation with 1ng/mL bFGF concentration. Fibronectin and collagen I expressions were higher after 24-h stimulation with 5ng/mL bFGF.
dBMMSCs (1ng/mL-bFGF stimulus after 24h) are very similar to dPLFs as regards morphological and immunostaining characteristics, and collagen and/or fibronectin production. The dBMMSCs presented the highest protein expression rates with 5ng/mL-bFGF stimulus after 24-h.
Full-text · Article · Mar 2014 · Archives of oral biology
[Show abstract][Hide abstract] ABSTRACT: Tendinous lesions are very common in athlete horses. The process of tendon healing is slow and the quality of the new tissue is often inferior to the original, leading in many cases to recurrence of the lesion. One of the main reasons for the limited healing capacity of tendons is its poor vascularization. At present, cell therapy is used in equine practice for the treatment of several disorders including tendinitis, desmitis and joint disease. However, there is little information regarding the mechanisms of action of these cells during tissue repair. It is known that Mesenchymal Stem Cells (MSCs) release several growth factors at the site of implantation, some of which promote angiogenesis. Comparison of blood flow using power Doppler ultrasonography was performed after the induction superficial digital flexor tendon tendinitis and implantation of adipose tissue-derived MSCs in order to analyze the effect of cell therapy on tendon neovascularization. For quantification of blood vessel histopathological examinations were conducted. Increased blood flow and number of vessels was observed in treated tendons up to 30 days after cell implantation, suggesting promotion of angiogenesis by the cell therapy.
[Show abstract][Hide abstract] ABSTRACT: The use of the 5-alpha reductase inhibitors (5-ARIs) finasteride and dutasteride for prostate cancer prevention is still under debate. The FDA recently concluded that the increased prevalence of high-grade tumors among 5-ARI-treated patients must not be neglected, and they decided to disallow the use of 5-ARIs for prostate cancer prevention. This study was conducted to verify the effects of finasteride on prostate cell migration and invasion and the related enzymes/proteins in normal human and tumoral prostatic cell lines.
RWPE-1, LNCaP, PC3 and DU145 cells were cultivated to 60% confluence and exposed for different periods to either 10 µM or 50 µM finasteride that was diluted in culture medium. The conditioned media were collected and concentrated, and MMP2 and MMP9 activities and TIMP-1 and TIMP-2 protein expression were determined. Cell viability, migration and invasion were analyzed, and the remaining cell extracts were submitted to androgen receptor (AR) detection by western blotting techniques. Experiments were carried out in triplicate.
Cell viability was not significantly affected by finasteride exposure. Finasteride significantly downregulated MMP2 and MMP9 activities in RWPE-1 and PC3 cells and MMP2 in DU145 cells. TIMP-2 expression in RWPE-1 cells was upregulated after exposure. The cell invasion of all four tested cell lines was inhibited by exposure to 50 µM of finasteride, and migration inhibition only occurred for RWPE-1 and LNCaP cells. AR was expressed by LNCaP, RWPE-1 and PC3 cells.
Although the debate on the higher incidence of high-grade prostate cancer among 5-ARI-treated patients remains, our findings indicate that finasteride may attenuate tumor aggressiveness and invasion, which could vary depending on the androgen responsiveness of a patient's prostate cells.
[Show abstract][Hide abstract] ABSTRACT: The purpose of this study was to evaluate the effect of intralesional Mesenchymal Stem Cells (MSC) on the treatment of experimentally induced articular chondral defects in horses, emphasizing the benefits of this application in veterinary medicine. Chondral defects were induced in the medial femoral trochlea of both hind limbs of four horses. Thirty days post induction; the horses were divided into two groups. The G1 was submitted to treatment with MSC and the G2 was the control group. Clinical evaluations, synovial fluid analysis and synovial Prostaglandin E 2 (PGE 2) assessment were performed prior to defects and fortnightly up to 120 and 150 days. Macroscopic, histopathological and histochemical evaluations were performed at the end of the experiment. The treatment with MSC reduced the intraarticular inflammatory process. The G1 showed lower PGE 2 concentrations in the synovial fluid and greater percentage of mononuclear cells and lower percentages of lymphocytes and neutrophils. The treatment improved the macro and microscopic aspects of repair tissue. No difference was observed in the scores of lameness between the G1 and G2. The use of MSC in the treatment of chondral defects minimized joint inflammation, as confirmed by synovial fluid analysis. The treatment resulted in an improved repair tissue, verified by macroscopic examination, histochemical and histopathological analysis.
Full-text · Article · Oct 2013 · Stem Cell Discovery
[Show abstract][Hide abstract] ABSTRACT: Platelet-rich plasma has been largely used as a therapeutic option for the treatment of chronic wounds of different etiologies. The enhanced regeneration observed after the use of platelet-rich plasma has been systematically attributed to the growth factors that are present inside platelets' granules.
We hypothesize that the remaining plasma and platelet-bound fibronectin may act as a further bioactive protein in platelet-rich plasma preparations.
Recent reports were analyzed and presented as direct evidences of this hypotheses.
Fibronectin may directly influence the extracellular matrix remodeling during wound repair. This effect is probably through matrix metalloproteinase expression, thus exerting an extra effect on chronic wound regeneration.
Physicians should be well aware of the possible fibronectin-induced effects in their future endeavors with PRP in chronic wound treatment.
[Show abstract][Hide abstract] ABSTRACT: Stem cell therapy has shown promising results in tendinitis and osteoarthritis in equine medicine. The purpose of this work was to characterize the adipose-derived mesenchymal stem cells (AdMSCs) in horses through (1) the assessment of the capacity of progenitor cells to perform adipogenic, osteogenic and chondrogenic differentiation; and (2) flow cytometry analysis using the stemness related markers: CD44, CD90, CD105 and MHC Class II. Five mixed-breed horses, aged 2-4 years-old were used to collect adipose tissue from the base of the tail. After isolation and culture of AdMSCs, immunophenotypic characterization was performed through flow cytometry. There was a high expression of CD44, CD90 and CD105, and no expression of MHC Class II markers. The tri-lineage differentiation was confirmed by specific staining: adipogenic (Oil Red O), osteogenic (Alizarin Red), and chondrogenic (Alcian Blue). The equine AdMSCs are a promising type of adult progenitor cell for tissue engineering in veterinary medicine.
Full-text · Article · Aug 2013 · Arquivo Brasileiro de Medicina Veterinária e Zootecnia
[Show abstract][Hide abstract] ABSTRACT: Tendon injury is a major cause of lameness and decreased performance in athletic equines. Various therapies for tendonitis have been described; however, none of these therapies results in complete tissue regeneration, and the injury recurrence rate is high even after long recovery periods involving rest and physiotherapy.
A lesion was induced with collagenase gel in the superficial digital flexor tendon in the center portion of the metacarpal region of eight equines of mixed breed. After two weeks, the lesions of the animals in the treated and control groups were treated through the intralesional administration of mesenchymal stem cells derived from adipose tissue (adMSCs) suspended in platelet concentrate (PC) and with phosphate buffered saline (PBS), respectively. Serial ultrasound analyses were performed every two weeks. After 16 weeks of therapy, a biopsy was performed for histopathological, immunohistochemical, and gene expression (type I collagen (COL1A1), type III collagen (COL3A1), tenascin-C (TNC), tenomodulin (TNMD), and scleraxis (SCX)) analyses.
Differences in the ultrasound and histopathological analyses were observed between the groups. Improved results were reported in the group treated with adMSCs suspended in PC. There was no difference in the gene expression levels observed after the different treatments. The main results observed from the histopathological evaluation of the treated group were as follows: a prevention of the progression of the lesion, a greater organization of collagen fibers, and a decreased inflammatory infiltrate. A lack of progression of the lesion area and its percentage was observed in the ultrasound, and increased blood flow was measured by Power Doppler.
The use of adMSCs combined with PC for the therapy of experimentally induced tendonitis prevented the progression of the tendon lesion, as observed in the ultrasound examination, and resulted in a greater organization and decreased inflammation, as observed in the histopathological evaluation. These data demonstrate the therapeutic potential of this therapy for the treatment of equine tendonitis.
[Show abstract][Hide abstract] ABSTRACT: Cardiovascular diseases remain leaders as the major causes of mortality in Western society. Restoration of the circulation through construction of bypass surgical treatment is regarded as the gold standard treatment of peripheral vascular diseases, and grafts are necessary for this purpose. The great saphenous vein is often not available and synthetic grafts have their limitations. Therefore, new techniques to produce alternative grafts have been developed and, in this sense, tissue engineering is a promising alternative to provide biocompatible grafts. This study objective was to reconstruct the endothelium layer of decellularized vein scaffolds, using mesenchymal stem cells (MSCs) and growth factors obtained from platelets.
Fifteen nonpregnant female adult rabbits were used for all experiments. Adipose tissue and vena cava were obtained and subjected to MSCs isolation and tissue decellularization, respectively. MSCs were subjected to differentiation using endothelial inductor growth factor (EIGF) obtained from human platelet lysates. Immunofluorescence, histological and immunohistochemical analyses were employed for the final characterization of the obtained blood vessel substitute.
The scaffolds were successfully decellularized with sodium dodecyl sulfate. MSCs actively adhered at the scaffolds, and through stimulation with EIGF were differentiated into functional endothelial cells, secreting significantly higher quantities of von Willebrand factor (0.85 μg/mL; P < .05) than cells cultivated under the same conditions, without EIGF (0.085 μg/mL). Cells with evident morphologic characteristics of endothelium were seen at the lumen of the scaffolds. These cells also stained positive for fascin protein, which is highly expressed by differentiated endothelial cells.
Taken together, the use of decellularized bioscaffold and subcutaneous MSCs seems to be a potential approach to obtain bioengineered blood vessels, in the presence of EIGF supplementation.
Full-text · Article · Jul 2013 · Journal of vascular surgery: official publication, the Society for Vascular Surgery [and] International Society for Cardiovascular Surgery, North American Chapter
[Show abstract][Hide abstract] ABSTRACT: A new highly luminescent europium complex with formula [Eu(TTA)3(Bpy-Si)] where TTA stands for the thenoyltrifluoroacetone, (C4H3S)COCH2COCF3, chelating ligand and Bpy-Si, Bpy-CH2NH(CH2)3Si(OEt)3, is an organosilyldipyridine ligand displaying a triethoxysilyl group as grafting function, has been synthesized and fully characterized. This bi-functional complex has been grafted on the surface of dense silica nanoparticles (NPs) and on mesoporous silica microparticles as well. The covalent bonding of [Eu(TTA)3(Bpy-Si)] inside uniform Stöber silica nanoparticles was also achieved. The general methodology proposed could be applied to any silica matrix, allowed high grafting ratios overcoming chelate release and tendency to agglomeration. Luminescent silica-based nanoparticles SiO2-[Eu(TTA)3(Bpy-Si)], with diameter of 28 +- 2 nm, were successfully tested as luminescent labels for the imaging of Pseudomonas aeruginosa biofilms. They were also functionalized by a specific monoclonal antibody and subsequently employed for the selective imaging of Escherichia coli bacteria.