[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.
Clinics (São Paulo, Brazil) 12/2014; 69(10):694-8. · 1.59 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: 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.
Clinics (São Paulo, Brazil) 07/2014; 69(7):500-503. · 1.59 Impact Factor
[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.
[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.
[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.
Archives of oral biology 03/2014; 59(3):268-76. · 1.65 Impact Factor
[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.
[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.
[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.
Arquivo Brasileiro de Medicina Veterinária e Zootecnia 08/2013; 65(4):939-945. · 0.27 Impact Factor
[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.
Journal of vascular surgery: official publication, the Society for Vascular Surgery [and] International Society for Cardiovascular Surgery, North American Chapter 07/2013; · 3.52 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: To study the effect of isoflurane (Iso) or propofol (Prop) anesthesia on renal ischemia/reperfusion injury (IRI) during transient hyperglycemia.
Thirty six rats were randomly assigned into six groups of six animals each: PHS (Sham-Prop=1mg.kg-1.min-1 + Hyperglycemia=2.5g.kg-1 of glucose solution administered intraperitoneally); HIS (Sham-Iso + Hyperglycemia); PHI (Prop + Hyperglycemia + Ischemia); IHI (Iso + Hyperglycemia + Ischemia); PI (Prop + Ischemia), and II (Iso + Ischemia). After 30 minutes of anesthesia induction, right nephrectomy was performed (all animals) and the left renal artery was clamped during 25 minutes (ischemia). The animals were sacrificed after 24 hours and blood collection (to dose creatinine) and left kidney removal were performed for histological analysis, and flow cytometry (FCM): percentage of initial apoptosis (APTi) and viable cells (VC).
Serum creatinine (mg/dL) was statistically different in groups PHI (3.60±0.40) and IHI (3.23±1.08), p<0.05. Histological analysis was statistically different in groups PHI (4.0[4.0;5.0]) and IHI (4.5[4.0;5.0]), p<0.05. APTi percentage was statistically different in groups PHI (73.2±7.1), and IHI (48.1±14). VC percentage was statistically different in groups PHI (25.8±6.9) and IHI (38.5±9.2), p<0.05.
Propofol and isoflurane showed the same level of protection against ischemia/reperfusion injury in the normoglycemic groups. Transient hyperglycemia is associated with an increase in IRI.
Acta cirurgica brasileira / Sociedade Brasileira para Desenvolvimento Pesquisa em Cirurgia 03/2013; 28(3):161-6. · 0.48 Impact Factor
[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.
PLoS ONE 01/2013; 8(12):e84757. · 3.53 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: High-grade prostate cancers express high levels of matrix metalloproteinases (MMPs), major enzymes involved in tumor invasion and metastasis. However, the commonly employed tumoral cell lines on prostate cancer research express small amounts of MMPs when cultivated in monolayer cultures, with common culture media. The present study was conducted to verify if culture conditions with fibronectin alters MMP2 and MMP9 expression by human prostatic epithelial cell lines RWPE-1, LNCaP and PC-3. These cells were individually seeded at 2x10(4) cels/cm(2), cultivated until 80% confluence, and further exposed for 4 hours to fibronectin, after which the conditioned medium was analyzed by gelatin zymography. Untreated cells were given common medium. Only RWPE-1 express detectable amounts of MMP9 when cultivated in common medium, while addition of fibronectin induced high expression of pro and active forms of MMP2 in all tested cell lines. Our findings demonstrate that normal and tumoral prostate cell lines express MMP2 activity when in contact with extracellular matrix components or blood plasma proteins such as fibronectin. Future studies about transcriptomes and proteomes in prostate cancer research using these cell lines should not neglect these important conclusions.
Biochemical and Biophysical Research Communications 12/2012; · 2.28 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Articular lesions are still a major challenge in orthopedics because of cartilage's poor healing properties. A major improvement in therapeutics was the development of autologous chondrocytes implantation (ACI), a biotechnology-derived technique that delivers healthy autologous chondrocytes after in vitro expansion. To obtain cartilage-like tissue, 3D scaffolds are essential to maintain chondrocyte differentiated status. Currently, bioactive 3D scaffolds are promising as they can deliver growth factors, cytokines, and hormones to the cells, giving them a boost to attach, proliferate, induce protein synthesis, and differentiate. Using mesenchymal stem cells (MSCs) differentiated into chondrocytes, one can avoid cartilage harvesting. Thus, we investigated the potential use of a platelet-lysate-based 3D bioactive scaffold to support chondrogenic differentiation and maintenance of MSCs. The MSCs from adult rabbit bone marrow (n = 5) were cultivated and characterized using three antibodies by flow cytometry. MSCs (1 × 10(5)) were than encapsulated inside 60 µl of a rabbit platelet-lysate clot scaffold and maintained in Dulbecco's Modified Eagle Medium Nutrient Mixture F-12 supplemented with chondrogenic inductors. After 21 days, the MSCs-seeded scaffolds were processed for histological analysis and stained with toluidine blue. This scaffold was able to maintain round-shaped cells, typical chondrocyte metachromatic extracellular matrix deposition, and isogenous group formation. Cells accumulated inside lacunae and cytoplasm lipid droplets were other observed typical chondrocyte features. In conclusion, the usage of a platelet-lysate bioactive scaffold, associated with a suitable chondrogenic culture medium, supports MSCs chondrogenesis. As such, it offers an alternative tool for cartilage engineering research and ACI.
[Show abstract][Hide abstract] ABSTRACT: Hyperglycemia is associated with a decreased tolerance to ischemia and an increased severity of renal ischemia reperfusion (I/R) injury. It has been suggested that erythropoietin (EPO) attenuates this effect in normoglycemic animals. This study sought to examine the effects of EPO on treatment renal I/R injury (IRI) in transiently hyperglycemic rats.
Twenty-eight male Wister rats anesthetized with isoflurane received glucose (2.5 g.kg(-1) intraperitoneally) before right nephrectomy. They were randomly assigned to four groups: sham operation (S); IRI (ISO); IRI+EPO, (600 UI kg(-1) low-dose EPO [EL]); and IRI+EPO 5000 UI kg(-1) (high-dose EPO [EH]). IRI was induced by a 25-minute period of left renal ischemia followed by reperfusion for 24 hours. Serum creatinine and glucose levels were measure at baseline (M1), immediately after the ischemic period (M2), and at 24 hours after reperfusion (M3). After sacrificing the animals, left kidney specimens were submitted for histological analysis including flow cytometry to estimate tubular necrosis and the percentages of apoptotic, dead or intact cells.
Scr in the ISO group was significantly higher at M3 than among the other groups. Percentages of early apoptotic cells in ISO group were significantly higher than the other groups. Percentages of late apoptotic cells in S and ISO groups were significantly greater than EL and EH groups. However, no significant intergroup differences were observed regarding the incidence of tubular necrosis.
Our results suggested that, although not preventing the occurrence of tubular necrosis, EPO attenuated apoptosis and glomerular functional impairment among transiently hyperglycemic rats undergoing an ischemia/reperfusion insult.
[Show abstract][Hide abstract] ABSTRACT: Superficial digital flexor tendon lesion is an important cause of lameness in equine
athletes. Although numerous treatments have been described, few are effective at
promoting significant improvement in the quality of the extracellular matrix. Therefore,
great potential remains for recurrence and in certain cases, an abrupt end to the horse’s
athletic career. Recently, several experiments have focused on the therapeutic potential
of mesenchymal stem cells (MSCs) in cases of tendon lesions. This study aimed to
evaluate the effect of adipose tissue-derived MSCs in the treatment of induced tendinitis
of the superficial digital flexor tendon in horses by clinical, ultrasonographic, histopathological,
and immunochemical analyses. Tendinitis was induced in both thoracic
limbs of eight mares by administration of collagenase solution and adipose tissue was
collected from the tail base for MSCs isolation and expansion, which were used during
cellular therapy on only one limb 30 days after lesion induction. No differences occurred
between the groups regarding the clinical and ultrasonographic analyses; however,
histopathological evaluation revealed a significant improvement in tendon fiber organization
and diminished inflammatory infiltrate, whereas immunohistochemical analysis
showed increased expression of type I collagen in the treated group as compared with
controls. The cellular therapy model implanted in this experiment promoted increased
perivascular inflammatory infiltrate, fibroblastic density, neovascularization, and qualitative
healing improvement of tendon extracellular matrix, in terms of fiber orientation
and type I/III collagen ratio; moreover, it was considered to be a safe and viable process.
[Show abstract][Hide abstract] ABSTRACT: Cysticercosis is a major cause of economic loss in bovine production due to meat condemnation. Chemotherapy is being used in Brazilian cattle and a diagnostic test to improve the treatment program is desired. We produced monoclonal antibodies against crude (TAEB) and cyst fluid (TAEF) Taenia saginata metacestode antigens using immunized BALB/c mice. After cell fusion, 10 TAEB and nine TAEF hybrids were selected and cloned resulting in 18 IgG(1) and 32 IgM TAEB clones, and 9 IgG(1) and 9 IgM TAEF clones. Ascites was produced and Western blot testing was performed resulting in reactivity to protein fractions of low molecular weight (<18kDa), 43, 55, 66 and 100kDa. The indirect immunofluorescence test, with one monoclonal antibody against crude and one against cyst fluid antigens, recognized antigenic fractions of both the scolex and the bladder wall of metacestodes from naturally infected bovine.