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PURPOSE: To examine the effect of various biologic adjuvants on the polarization of macrophages in an in vitro model for rotator cuff tears. METHODS: Tissue was harvested from 6 patients undergoing arthroscopic rotator cuff repair. An in vitro model of the supraspinatus and subacromial bursa was created and treated with control, platelet-rich plasma (PRP), autologous activated serum (AAS), or a combination of PRP+AAS. The effect of treatment on macrophage polarization between M1 proinflammatory macrophages or M2 anti-inflammatory macrophages was measured using gene expression, protein expression, flow cytometry, and nitric oxide production. RESULTS: Tendon and bursa treated with PRP, AAS, and PRP+AAS significantly decreased the gene expression of M1 markers interleukin (IL)-12 and tumor necrosis factor-alpha while significantly increasing the expression of M2 markers arginase, IL-10, and transforming growth factor-ß (P < .05) compared with treatment with control. Enzyme-linked immunosorbent assay analysis of protein production demonstrated that, compared with control, coculture treated with PRP, AAS, and PRP+AAS significantly decreased markers of M1-macrophages (IL-6, IL-12, and tumor necrosis factor-alpha) while significantly increasing the expression of markers of M2-macrophages (arginase, IL-10, and transforming growth factor-beta) (P < .05). Flow cytometry analysis of surface markers demonstrated that compared with control, tendon and bursa treated with PRP, AAS, and PRP+AAS significantly decreased markers of M1-macrophages (CD80, CD86, CD64, CD16) while significantly increasing the expression of markers of M2-macrophages (CD163 and CD206) (P < .05). Treatment of the coculture with PRP, AAS, and PRP+AAS consistently demonstrated a decrease in nitric oxide production (P < .05) compared with control. AAS and PRP+AAS demonstrated an increased macrophage shift to M2 compared with PRP alone, whereas there was not as uniform of a shift when comparing PRP+AAS with AAS alone. CONCLUSIONS: In an in vitro model of rotator cuff tears, the treatment of supraspinatus tendon and subacromial bursa with PRP, AAS, and PRP+AAS demonstrated an increase in markers of anti-inflammatory M2-macrophages and a concomitant decrease in markers of proinflammatory M1-macrophages. AAS and PRP+AAS contributed to a large shift to macrophage polarization to the anti-inflammatory M2 compared with PRP. CLINICAL RELEVANCE: The mechanism of biologic adjuvant effects on the rotator cuff remains poorly understood. This study suggests that they may contribute to polarization of macrophages for their proinflammatory (M1) state to the anti-inflammatory (M2) state.
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PURPOSE: To quantify cellular senescence in supraspinatus tendon and subacromial bursa of humans with rotator cuff tears and to investigate the in vitro efficacy of the senolytic dasatinib + quercetin (D+Q) to eliminate senescent cells and alter tenogenic differentiation. METHODS: Tissue was harvested from 41 patients (mean age, 62 years) undergoing arthroscopic rotator cuff repairs. In part 1 (n = 35), senescence was quantified using immunohistochemistry and gene expression for senescent cell markers (p16 and p21) and the senescence-associated secretory phenotype (SASP) (interleukin [IL] 6, IL-8, matrix metalloproteinase [MMP] 3, monocyte chemoattractant protein [MCP] 1). Senescence was compared between patients <60 and ≥60 years old. In part 2 (n = 6) , an in vitro model of rotator cuff tears was treated with D+Q or control. D+Q, a chemotherapeutic and plant flavanol, respectively, kill senescent cells. Gene expression analysis assessed the ability of D+Q to kill senescent cells and alter markers of tenogenic differentiation. RESULTS: Part 1 revealed an age-dependent significant increase in the relative expression of p21, IL-6, and IL-8 in tendon and p21, p16, IL-6, IL-8, and MMP-3 in bursa (P < .05). A significant increase was seen in immunohistochemical staining of bursa p21 (P = .028). In part 2, D+Q significantly decreased expression of p21, IL-6, and IL-8 in tendon and p21 and IL-8 in bursa (P < .05). Enzyme-linked immunosorbent assay analysis showed decreased release of the SASP (IL-6, MMP-3, MCP-1; P = .002, P = .024, P < .001, respectively). Tendon (P = .022) and bursa (P = .027) treated with D+Q increased the expression of COL1A1. CONCLUSIONS: While there was an age-dependent increase in markers of cellular senescence, this relationship was not consistently seen across all markers and tissues. Dasatinib + quercetin had moderate efficacy in decreasing senescence in these tissues and increasing COL1A1 expression. CLINICAL RELEVANCE: This study reveals that cellular senescence may be a therapeutic target to alter the biological aging of rotator cuffs and identifies D+Q as a potential therapy.
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Lesiones del Manguito de los Rotadores , Humanos , Persona de Mediana Edad , Lesiones del Manguito de los Rotadores/tratamiento farmacológico , Lesiones del Manguito de los Rotadores/cirugía , Dasatinib/farmacología , Dasatinib/uso terapéutico , Quercetina/farmacología , Quercetina/uso terapéutico , Metaloproteinasa 3 de la Matriz/genética , Interleucina-6/metabolismo , Interleucina-8 , Senescencia CelularRESUMEN
Purpose: To report the clinical outcomes after biologically augmented rotator cuff repair (RCR) with a fibrin scaffold derived from autologous whole blood and supplemented with concentrated bone marrow aspirate (cBMA) harvested at the proximal humerus. Methods: Patients who underwent arthroscopic RCR with biologic augmentation using a fibrin clot scaffold ("Mega- Clot") containing progenitor cells and growth factors from proximal humerus BMA and autologous whole blood between April 2015 and January 2018 were prospectively followed. Only high-risk patients in primary and revision cases that possessed relevant comorbidities or physically demanding occupation were included. Minimum follow-up for inclusion was 1 year. The visual analog score for pain (VAS), American Shoulder and Elbow Surgeons (ASES), Simple Shoulder Test (SST), Single Assessment Numerical Evaluation (SANE), and Constant-Murley scores were collected preoperatively and at final follow-up. In vitro analyses of the cBMA and fibrin clot using nucleated cell count, colony forming units, and live/dead assays were used to quantify the substrates. Results: Thirteen patients (56.9 ± 7.7 years) were included. The mean follow-up was 26.9 ± 17.7 months (n = 13). There were significant improvements in all outcome scores from the preoperative to the postoperative state: VAS (5.6 ± 2.5 to 3.1 ± 3.2; P < .001), ASES (42.0 ± 17.1 to 65.5 ± 30.6; P < .001), SST (3.2 ± 2.8 to 6.5 ± 4.7; P = .002), SANE (11.5 ± 15.6 to 50.3 ± 36.5; P < .001), and Constant-Murley (38.9 ± 17.5 to 58.1 ± 26.3; P < .001). Six patients (46%) had retears on postoperative MRI, despite all having improvements in pain and function except one. All failures were chronic rotator cuff tears, and all were revision cases except one (1.6 ± 0.5 previous RCRs). The representative sample of harvested cBMA showed an average of 28.5 ± 9.1 × 106 nucleated cells per mL. Conclusions: Arthroscopic rotator cuff repairs that are biologically augmented with a fibrin scaffold containing growth factors and autologous progenitor cells derived from autologous whole blood and humeral cBMA can improve clinical outcomes in primary, as well as revision cases in high-risk patients. However, the incidence of retears remains a concern in this population, demanding further improvements in biologic augmentation. Level of Evidence: IV, therapeutic case series.
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PURPOSE: The purpose was to evaluate the response of human ligamentocytes and osteoblasts after biological augmentation with thrombin, concentrated bone marrow aspirate (cBMA), or platelet-rich plasma (PRP) on two different types of nonresorbable flat braided suture used for ligament bracing. METHODS: Uncoated (U) and collagen-coated (C) flat braided suture material was augmented with either thrombin (T), cBMA (B), PRP (P), or a combination of these three (A), while platelet-poor plasma was used as a source for fibrin (F) in each assay. Previously cultured ligamentocytes and osteoblasts were added with a defined density and assayed after the required time period for adhesion, proliferation, and alkaline phosphatase activity. RESULTS: Biological augmentation of uncoated [(UFT, UFBT, UFA; P < .001), (UFPT; P = .017)] and collagen-coated suture (CFT, CFPT, CFBT, CFA; P < .001) led to a significantly higher ligamentocyte adhesion. Significantly higher adhesion was also observed for osteoblasts (UFT, UFPT, UFBT, UFA; P < .001; CFT, CFPT, CFBT, CFA; P < .001). Similarly, ligamentocyte proliferation was significantly higher [(UFT, UFPT, UFA; P = .009), (UFBT; P = .001), (CFT; P = .009), (CFBT; P = .001), and (CFA; P = .01)]. Osteoblasts showed significantly higher proliferation as well [(UFT, UFPT, UFA; P = .002), (UFBT; P = .001); (CFT: P = .003), and (CFPT, CFBT, CFA; P = .001)]. Augmentation with thrombin, PRP, and BMA for uncoated (UFT; P = .006, UFPT; P = .035, UFBT; P = .001) and BMA for coated suture (CFBT; P = .027) led to significantly higher alkaline phosphatase activity. CONCLUSION: Biological enhancement of suture used for ligament bracing significantly increased ligamentocyte and osteoblast adhesion and proliferation, as well as alkaline phosphatase activity of osteoblasts in an in vitro model. After biological augmentation, cellular adhesion, proliferation, and alkaline phosphatase activity changed up to 1,077%, 190%, and 78%, respectively. Furthermore, no overall superiority between uncoated or collagen-coated suture material was observed for cellular adhesion, proliferation, or alkaline phosphatase activity. CLINICAL RELEVANCE: This study provides in vitro data on a new treatment concept of biologic augmentation for acute ligamentous lesions treated with ligament bracing that has not been widely described. This concept may improve the healing of injured ligaments, in addition to providing immediate biomechanical stabilization.
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Osteoblastos , Plasma Rico en Plaquetas , Adhesión Celular , Proliferación Celular , Humanos , Ligamentos , Osteoblastos/fisiología , SuturasRESUMEN
PURPOSE/AIM: The purpose of this study is to identify a cell population within the murine subcromial bursal-derived cells with characteristics compatible to an accepted mesenchymal stem cell description given by the International Society for Cellular Therapy (ISCT). MATERIALS AND METHODS: Murine subacromial bursa was harvested using microsurgical technique. Subacromial bursal-derived cells were classified through colony-forming units, microscopic morphology, fluorescent-activated cell sorting, and differentiation into chondrogenic, adipogenic, and osteogenic lineages. RESULTS: Subacromial bursal samples exhibited cell growth out of the tissue for an average of 115 ± 29 colony-forming units per 1 mL of complete media. Subacromial bursal-derived cells exhibited a long, spindle-shaped, fibroblast-like morphology. Subacromial bursal-derived cells positively expressed mesenchymal stem cell markers CD73, CD90, and CD105, and negatively expressed mesenchymal stem cell markers CD31 and CD45. Subacromial bursal-derived cells, examined by Image J analysis and quantitative gene expression, were found to differentiate into chondrogenic, adipogenic, and osteogenic lineages. CONCLUSIONS: This study demonstrated the feasibility of harvesting murine subacromial bursal tissue and identified a cell population within the subacromial bursa with characteristics compatible to an accepted mesenchymal stem cell description. The results of this study suggest that the mouse subacromial bursal-derived cell population harbors mesenchymal stem cells. Murine subacromial bursal tissue is a potential source for obtaining cells with mesenchymal stem cell characteristics for future utilization in orthopedic research to look into treatment of rotator cuff pathology.
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Células Madre Mesenquimatosas , Lesiones del Manguito de los Rotadores , Articulación del Hombro , Animales , Bolsa Sinovial/patología , Diferenciación Celular , Ratones , Manguito de los Rotadores/patología , Lesiones del Manguito de los Rotadores/patologíaRESUMEN
Unsatisfactory failure rates following rotator cuff (RC) repair have led orthopaedic surgeons to explore biological augmentation of the healing enthesis. The subacromial bursa (SB) contains abundant connective tissue progenitor cells (CTPs) that may aid in this process. The purpose of the study was to investigate the influence of patient demographics and tear characteristics on the number of colony-forming units (CFUs) and nucleated cell count (NCC) of SB-derived CTPs. In this study, we harvested SB tissue over the supraspinatus tendon and muscle in 19 patients during arthroscopic RC repair. NCC of each sample was analyzed on the day of the procedure. After 14 days, CFUs were evaluated under a microscope. Spearman's rank correlation coefficient was then used to determine the relationship between CFUs or NCC and patient demographics or tear characteristics. The study found no significant correlation between patient demographics and the number of CFUs or NCC of CTPs derived from the SB (p > 0.05). The study did significantly observe that increased tear size was negatively correlated with the number of CFUs (p < 0.05). These results indicated that increased tear size, but not patient demographics, may influence the viability of CTPs and should be considered when augmenting RCrepairs with SB.
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PURPOSE: To compare the cellular viability and differentiation potential of subacromial bursa-derived cells (SBDCs) located over the rotator cuff muscle and tendon of patients undergoing primary versus revision arthroscopic rotator cuff repair (ARCR). METHODS: Subacromial bursa was harvested from 18 primary (57.1 ± 4.6 years) and 12 revision ARCRs (57.3 ± 6.7 years). Bursa was collected from 2 sites (over rotator cuff tendon and muscle), digested with collagenase, and grown in culture. The number of nucleated cells, colony-forming units (CFUs), differentiation potential, and mesenchymal stem cell surface markers were compared in primary and revision cases. RESULTS: There was no difference in the number of nucleated cells between primary and revision ARCR harvested from the subacromial bursa overlying the tendon (3019.3 ± 1420.6 cells/mg and 3541.7 ± 2244.2 cells/mg, respectively; P = .912) or muscle (2753.5 ± 1547.1 cells/mg and 2989.0 ± 2231.4 cells/mg, respectively; P = .777). There was no difference in the number of CFUs between primary and revision ARCR over the rotator cuff tendon (81.5 ± 49.5 CFUs and 53.0 ± 36.9 CFUs, respectively; P = .138), but there were significantly fewer CFUs over the muscle in revision cases (28.1 ± 22.7 CFUs) compared with primary cases (55.7 ± 34.5 CFUs) (P = .031). SBDCs from revision ARCR expressed characteristic mesenchymal stem cell surface epitopes and had multidifferentiation potentials for chondrogenesis, osteogenesis, and adipogenesis. CONCLUSIONS: SBDCs harvested over the rotator cuff muscle demonstrated significantly decreased colony-forming abilities in revision arthroscopic rotator cuff repairs compared with primary repairs. However, the subacromial bursa retains its pluripotent differentiation potential for chondrogenic, osteogenic, and adipogenic lineages in the revision setting. CLINICAL RELEVANCE: The subacromial bursa may play a role in the healing response of the repaired rotator cuff. This capacity is not necessarily diminished in the revision setting and may be harnessed as an orthobiologic.
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PURPOSE: To investigate the presence of connective tissue progenitor cells (CTPs) in the trochanteric bursa harvested over the gluteus medius muscle belly and tendon during open hip procedures. METHODS: Trochanteric bursa samples from nine patients (63.1 ± 8.6 years) undergoing total hip arthroplasty for primary osteoarthritis were obtained from 2 sites: over the gluteus medius tendon at the greater trochanter and over the muscle belly. Bursal tissue was digested with collagenase and grown in culture. The nucleated cell count, cellular concentration, cellular proliferation, fluorescence-activated cell sorting (FACS) analysis, and differentiation using immunostaining and quantitative polymerase chain reaction (PCR) were used to determine and quantify the presence of CTPs. RESULTS: Bursa-derived CTPs were identified in all harvested samples. At t = 0, there was no difference in nucleated cell count over muscle and tendon (1.69 ± 1.26 × 108 and 1.41 ± 1.12 × 108 cells/g, respectively; P = .162). Similarly, the cellular concentration at 3 weeks was not significantly different between bursa harvested over muscle and tendon (6.61 ± 5.14 × 106 and 5.58 ± 4.70 × 106 cells/g, respectively; P = .532). High cellular proliferation was identified for both bursal tissue overlying muscle and tendon (2.28 ± .95 and 1.66 ± 1.05, respectively; P = .194). FACS analysis revealed high positivity rates (>95%) of CTP-specific surface epitopes (CD105, CD90, and CD73) and low positivity rates (<1.3%) of negative markers (CD45, CD31). Osteogenic, adipogenic, and chondrogenic differentiation potential was demonstrated with immunostaining and quantitative PCR for gene expression. CONCLUSIONS: Connective tissue progenitor cells are found in the trochanteric bursa overlying the muscle and tendon of the hip abductors. CLINICAL RELEVANCE: During open hip procedures, the trochanteric bursa is often partially excised to identify muscular boundaries and tissue planes for surgical exposure. The function of the trochanteric bursa remains largely unknown. However, this tissue is a source of connective tissue progenitor cells, which may be important in the healing response of surgically repaired abductor tendons.
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PURPOSE: To evaluate the number and concentration of progenitors of the bone marrow aspirate (BMA) harvest from the body of the ilium in comparison with other established aspiration sites. METHODS: The inclusion criteria consisted of primary hip arthroscopy for acetabular labral tear. BMA was performed by placing an aspiration needle into the body of ilium just proximal to the sourcil in 33 patients. The BMA was centrifuged and processed in the operating room, resulting in approximately 3 to 5 mL of bone marrow aspirate concentrate (BMAC). Samples of both BMA and BMAC sample were analyzed. RESULTS: The cohort of 30 patients had a mean number of nucleated cells of 24.0 million nucleated cells/cc of BMA. The BMAC samples had a mean connective tissue progenitor (CTP) cell concentration of 879.3 stem cells/cc of BMAC, a mean CTP prevalence of 34.1 stem cells/million nucleated cells, and a mean number of days to form colonies of 2.97 days. All 4 metrics of CTP harvest did not vary significantly with age, body mass index, sex, or laterality. The nucleated cell count was significantly associated with both CTP prevalence, r2 = 0.287 (P = .002), and CTP concentration, r2 = 0.388 (P < .001). CONCLUSIONS: BMAC harvested from the body of the ilium during concurrent hip arthroscopy is a technically and biologically feasible option. Furthermore, the harvest site was found to have a CTP concentration that is similar or exceeds other published harvest sites. Finally, BMAC processing and application to areas of articular cartilage wear was performed efficiently and safely with no increase in morbidity or complications. CLINICAL RELEVANCE: The body of the ilium is a reliable and rich source of CTP cells. This study may assist orthopaedic surgeons interested in performing biologic augmentation during hip surgery in determining a harvest site.
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Acetábulo/cirugía , Artroscopía/métodos , Células de la Médula Ósea/citología , Enfermedades de los Cartílagos/cirugía , Cartílago Articular/cirugía , Células Madre/citología , Recolección de Tejidos y Órganos/métodos , Acetábulo/patología , Adulto , Enfermedades de los Cartílagos/patología , Cartílago Articular/patología , Recuento de Células , Femenino , Estudios de Seguimiento , Humanos , Ilion , Masculino , Persona de Mediana Edad , Estudios Prospectivos , Adulto JovenRESUMEN
PURPOSE: To identify an effective, nonenzymatic method for maximizing the yield of subacromial bursa-derived nucleated cells for augmenting rotator cuff repair. METHODS: Subacromial bursa (minimum 0.2 g) was collected prospectively over the supraspinatus from patients (n = 7) with at least one full-thickness tendon tear undergoing arthroscopic primary rotator cuff repair. Samples were processed and analyzed prospectively using 4 different methods: (1) mechanical digestion with scissors (chopping), (2) collagenase digestion, (3) mechanical digestion with a tissue homogenizer, and (4) whole tissue with minimal manipulation. Tissue from each method were plated and cultured in a low oxygen tension, humidified incubator for 7 days. Following incubation, cellularity was assessed with nucleated cell count using a Coulter Counter. Flow cytometry was performed on the non-enzymatic method that demonstrated the greatest cell count to confirm the presence of mesenchymal stem cells (MSCs). The Kruskal-Wallis H test and post hoc Dunn's test were used for statistical analysis. RESULTS: Following incubation, mean nucleated cell counts (cells/mL) were (1) 102,681 ± 73,249 for chopping, (2) 76,190 ± 66,275 for collagenase, (3) 31,686 ± 29,234 for homogenization, and (4) 11,162 ± 4016 for whole tissue. There was no significant difference between chopping and collagenase (P = .45) or between homogenization and collagenase (P = .52). Both chopping (P = .003) and collagenase (P = .03) produced significantly more cells when compared with whole tissue. Flow cytometry confirmed the presence of MSC markers on samples processed by chopping. CONCLUSIONS: Mechanical isolation of subacromial bursa-derived cells using a chopping technique demonstrated similar nucleated cell count compared with collagenase, along with the confirmed presence of MSCs. CLINICAL RELEVANCE: This study demonstrated a nonenzymatic, mechanical method for isolating subacromial bursa-derived cells to potentially augment rotator cuff repair. Further clinical studies are required to assess its possible advent in the tendon-bone healing process.
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Artroscopía/métodos , Bolsa Sinovial/cirugía , Células Madre Mesenquimatosas/citología , Procedimientos de Cirugía Plástica/métodos , Lesiones del Manguito de los Rotadores/cirugía , Manguito de los Rotadores/cirugía , Cicatrización de Heridas , Recuento de Células , Femenino , Humanos , Masculino , Persona de Mediana EdadRESUMEN
PURPOSE: To compare the potency of mesenchymal stem cells between the cells derived from the subacromial bursa to concentrated bone marrow aspirate (cBMA) taken from patients undergoing rotator cuff (RC) repair. METHODS: Subacromial bursa and cBMA were harvested arthroscopically from 13 patients (age 57.4 ± 5.2 years, mean ± standard deviation) undergoing arthroscopic primary RC repair. Bone marrow was aspirated from the proximal humerus and concentrated using an automated system (Angel System; Arthrex). Subacromial bursa was collected from 2 sites (over the RC tendon and muscle) and digested with collagenase to isolate a single cellular fraction. Proliferation, number of colony-forming units, differentiation potential, and gene expression were compared among the cells derived from each specimen. RESULTS: The cells derived from subacromial bursa showed significantly higher proliferation compared with the cells derived from cBMA after 5, 7, and 10 days (P = .018). Regarding colony-forming units, the subacromial bursa had significantly more colonies than cBMA (P = .002). Subacromial bursal cells over the RC tendon produced significantly more colonies than cells over both the RC muscle and cBMA (P = .033 and P = .028, respectively). Moreover, when compared with cBMA, cells derived from subacromial bursa showed significantly higher differentiation ability and higher gene expression indicative of chondrogenesis, osteogenesis, and adipogenesis. CONCLUSION: The subacromial bursa is an easily accessible tissue that can be obtained during RC repair, with significant pluripotent stem cell potency for tendon healing. Compared with cBMA taken from the proximal humerus, bursal cells showed significantly increased differentiation ability and gene expression over time. CLINICAL RELEVANCE: Failed RC repairs have been partly attributed to a poor healing environment. Biologic augmentation of the repair site may help increase healing potential and incorporation of the cuff at the tendon-bone interface.
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Artroscopía/métodos , Bolsa Sinovial/patología , Células Madre Mesenquimatosas/citología , Lesiones del Manguito de los Rotadores/cirugía , Manguito de los Rotadores/cirugía , Diferenciación Celular , Femenino , Humanos , Masculino , Persona de Mediana Edad , Manguito de los Rotadores/patología , Lesiones del Manguito de los Rotadores/diagnósticoRESUMEN
BACKGROUND: Numerous studies have identified factors that may affect the chances of rotator cuff healing after surgery. Intraoperative tendon quality may be used to predict healing and to determine type of repair and/or consideration of augmentation. There are no data that correlate how gross tendon morphology and degree of tendinopathy affect patient outcome or postoperative tendon healing. Purpose/Hypothesis: The purposes of this study were to (1) compare the gross appearance of the tendon edge during arthroscopic rotator cuff repair with its histological degree of tendinopathy and (2) determine if gross appearance correlated with postoperative repair integrity. The hypothesis was that gross (macroscopic) tendon with normal thickness, no delamination, and elastic tissue before repair would have a correlation with low Bonar scores, higher postoperative American Shoulder and Elbow Surgeons (ASES) scores, and increased rates of postoperative tendon healing on ultrasound. STUDY DESIGN: Cross-sectional study; Level of evidence, 3. METHODS: A total of 105 patients undergoing repair of medium-size (1-3 cm) full-thickness rotator cuff tears were enrolled in the study. Intraoperatively, the supraspinatus tendon was rated on thickness, fraying, and stiffness. Tendon tissue was recovered for histological analysis based on the Bonar scoring system. Postoperative ASES and ultrasound assessment of healing were obtained 1 year after repair. Correlation between gross appearance of the tendon and rotator cuff histology was determined. RESULTS: Of the 105 patients, 85 were followed the study to completion. The mean age of the patients was 61.6 years; Bonar score, 7.5; preoperative ASES score, 49; and postoperative ASES score, 86. Ninety-one percent of repairs were intact on ultrasound. Gross appearance of torn rotator cuff tendon tissue did not correlate with histological appearance. Neither histological (Bonar) score nor gross appearance correlated with multivariate analysis of ASES score, postoperative repair status, or demographic data. CONCLUSION: The degree of tendinopathy did not correlate with morphological appearance of the tendon. Neither of these parameters correlated with healing or patient outcome. This study suggests that the degree of tendinopathy, unlike muscle atrophy, may not be predictive of outcomes and that, on appearance, poor quality tendon has adequate healing capacity. Therefore, abnormal gross tendon appearance should not affect the repair effort or technique.
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Artroscopía/métodos , Lesiones del Manguito de los Rotadores/cirugía , Manguito de los Rotadores/cirugía , Tendinopatía/cirugía , Anciano , Estudios Transversales , Femenino , Humanos , Masculino , Persona de Mediana Edad , Atrofia Muscular/cirugía , Periodo Posoperatorio , Procedimientos de Cirugía Plástica , Articulación del Hombro/cirugía , Tendones/cirugía , Resultado del Tratamiento , UltrasonografíaRESUMEN
BACKGROUND: The use of corticosteroids and local anesthetics to treat osteoarthritis has established benefits, including relief of pain and increased range of motion, but may also have the potential to lead to tissue atrophy or degeneration, specifically on chondrocytes. There is growing evidence that platelet-rich plasma (PRP) has anti-inflammatory characteristics that can limit the cytotoxic effects of corticosteroids and local anesthetics. Hypothesis/Purpose: The purpose of this study was to determine the effects of PRP in chondrocyte cultures when combined with corticosteroids or local anesthetics. The hypothesis of this study was that PRP would (1) dampen the negative effects on chondrocyte viability and (2) improve chondrocyte proliferation seen with corticosteroid or local anesthetic treatment alone. STUDY DESIGN: Controlled laboratory study. METHODS: Peripheral blood was obtained from 8 healthy participants, followed by centrifugation to obtain PRP. Human chondrocytes were treated with PRP alone or in combination with corticosteroids or local anesthetics. Saline (concentration of 0.9%) served as the control. Luminescence and radioactive thymidine assays were performed to examine chondrocyte viability and proliferation, respectively. Cell exposures of 0, 5, 10, and 30 minutes were used for viability and 120 hours for proliferation. RESULTS: The presence of PRP significantly limited the negative effect on chondrocyte viability at tested time points for the examined corticosteroids and local anesthetics ( P < .05). PRP in addition to corticosteroids and local anesthetics significantly improved chondrocyte proliferation ( P < .05). CONCLUSION: The addition of PRP can significantly reduce the cytotoxic effects of corticosteroids and/or local anesthetics applied to chondrocytes. PRP can improve the proliferation of chondrocytes compared with corticosteroids or local anesthetics alone. CLINICAL RELEVANCE: With the use of corticosteroids and local anesthetics for temporary symptomatic relief and improvement of function to treat the chronic progressive nature of osteoarthritis, long-term negative effects of these agents can be limited with the parallel use of PRP.
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Corticoesteroides/uso terapéutico , Anestésicos Locales/uso terapéutico , Condrocitos/fisiología , Plasma Rico en Plaquetas/fisiología , Adulto , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Condrocitos/efectos de los fármacos , Humanos , Persona de Mediana Edad , Adulto JovenRESUMEN
BACKGROUND: Platelet-rich plasma (PRP) has anti-inflammatory effects with potential applications in the treatment of osteoarthritis (OA). PURPOSE: To use an in vitro coculture model of OA in human cartilage and synovium to investigate the anti-inflammatory effects of 2 different PRP preparations. STUDY DESIGN: Controlled laboratory study. METHODS: A coculture system was created using osteoarthritic cartilage and synovium from 9 patients undergoing total knee arthroplasty. Interleukin-1ß (IL-1ß) was added to each coculture to induce inflammation. Two PRP preparations were obtained-one yielding low white blood cell and platelet concentrations (PRPLP) and one yielding high platelet and white blood cell concentrations (PRPHP). Either PRPLP, PRPHP, or medium was added to the coculture wells. Control wells contained OA cartilage and synovium but neither IL-1ß nor PRP. Normal, non-OA cartilage was obtained to establish baseline gene expression levels. Quantitative polymerase chain reaction was used to measure changes in markers of inflammation in the tissues (a disintegrin and metalloproteinase with thrombospondin motifs-5 [ADAMTS-5], tissue inhibitor of metalloproteinases-1 [TIMP-1], vascular endothelial growth factor [VEGF], aggrecan, and type I collagen) at 0, 24, 48, and 72 hours. RESULTS: Treatment with PRPLP or PRPHP significantly decreased expression of TIMP-1 and ADAMTS-5 in cartilage, increased aggrecan expression in cartilage, and decreased ADAMTS-5, VEGF, and TIMP-1 expression in synovium compared with control cocultures (P < .05). There was significantly less nitric oxide production in the PRPLP and PRPHP groups compared with controls (P < .05). There were significant differences in gene expression in the normal cartilage compared with all 4 groups of OA cartilage at all 4 time points. Treatment with either PRPLP or PRPHP returned some gene expression to the same levels in normal cartilage but not for all markers of inflammation. CONCLUSION: This coculture model assessed 2 different PRP preparations and their anti-inflammatory effects over time on human OA cartilage and synovium. Both had a significant anti-inflammatory effect on gene expression; however, there was no difference in the anti-inflammatory effect between the 2 preparations. CLINICAL RELEVANCE: Osteoarthritis is a leading cause of chronic disability, and less invasive treatment methods are needed. Study results suggest that PRP injections may be an effective alternative anti-inflammatory agent in the treatment of OA.
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Antiinflamatorios/farmacología , Biomarcadores/metabolismo , Osteoartritis de la Rodilla/terapia , Plasma Rico en Plaquetas , Proteínas ADAM/metabolismo , Proteína ADAMTS5 , Adulto , Agrecanos/metabolismo , Estudios de Casos y Controles , Técnicas de Cocultivo , Colágeno Tipo I/metabolismo , Femenino , Expresión Génica , Humanos , Inflamación/inducido químicamente , Interleucina-1beta/toxicidad , Masculino , Persona de Mediana Edad , Óxido Nítrico/biosíntesis , Osteoartritis de la Rodilla/metabolismo , Membrana Sinovial/metabolismo , Inhibidor Tisular de Metaloproteinasa-1/metabolismo , Factor A de Crecimiento Endotelial Vascular/metabolismoRESUMEN
Current clinical application of platelet-rich plasma is showing a trend toward multiple treatments. The goal of this study was to show the benefit of interval platelet-rich plasma application in the healing and recovery of human tenocytes using an in vitro cell model. Eight volunteers (6 men and 2 women) were included in this study (mean±SD age, 31.6±10.9 years). Venous blood was collected from new blood draws at 3 different times. Two blood products were prepared on each day of treatment: platelet-rich plasma derived from a single-spin process (PRPSS) and platelet-rich plasma derived from a double-spin process (PRPDS). The study had 2 limbs: 2-day and 4-day intervals. Cell proliferation, measured as disintegrations per minute, was then examined via a radioactive thymidine assay. In the 2-day-interval group, the difference in disintegrations per minute between days 0 and 2 in the PRPSS group reached statistical significance (P =.006). In the PRPDS group, statistical difference was seen between days 0 and 4 (P=.001) and between days 2 and 4 (P=.030). In the 4-day-interval group, the difference in disintegrations per minute between days 4 and 8 in the PRPSS group reached statistical significance, showing a decrease in cell proliferation (P =.013). In the PRPDS group, a statistical difference was seen between days 0 and 8 (P=.021), also showing a decrease in cell proliferation. The greatest effect of platelet-rich plasma, which has a positive effect on tenocyte proliferation and recovery, is seen on initial application. Its effect is diminished with repetitive application, and this finding leads to questioning of the efficacy of interval platelet-rich plasma dosing.
Asunto(s)
Proliferación Celular/fisiología , Plasma Rico en Plaquetas , Tendones/citología , Adulto , Células Cultivadas , Femenino , Humanos , Masculino , Tendones/fisiología , Cicatrización de Heridas , Adulto JovenRESUMEN
INTRODUCTION: cell-based tissue engineering strategies using human mesenchymal stem cells (hMSCs) may help to augment tendon healing. To further investigate the in-vitro behavior of this cell population, we investigated low oxygen culture levels, and growth factor supplementation and their effect on expression of tendon extracellular proteins and cell proliferation. MATERIALS AND METHODS: bone marrow aspirate (BMA) was harvested during arthroscopic rotator cuff repair. Characterized hMSCs derived from BMA were incorporated into 3-dimensional tissue engineered constructs (TECs). TECs were analyzed by frozen sections with immunohistochemistry for cell density, collagen I and collagen III expression. RESULTS: growth factor stimulation and low oxygen increased cell density within TECs. Low oxygen and addition of growth factors to culture media demonstrated an increase in collagen I and III expression, both in ambient oxygen conditions and low oxygen conditions. CONCLUSION: low oxygen and TGFß3 demonstrated a positive effect on cell number, and type I and III collagen expression in 3D culture environments.
RESUMEN
PURPOSE: This study investigates the antibacterial properties of two different platelet-rich plasma (PRP) platelet concentration preparations (PRPLP and PRPHP) through a time-kill assay. METHODS: Two different PRP preparations - a single spin process yielding lower white blood cells and platelet concentration (PRPLP) and one yielding high platelet and white blood cell concentration (PRPHP) - were obtained from 2 individuals. PRPLP, PRPHP, phosphate buffered saline (PBS), whole blood and Cefazolin were added to experimental reaction tubes, each containing a single bacterial inoculum of Staphylococcus aureus (S. aureus), Staphylococcus epidermidis (S. epi), methicillin-resistant Staphylococcus aureus (MRSA) or Propionibacterium acnes (P. acnes). Two dilutions (1:1,000, and 1:10,000) were plated in duplicate tubes, along with positive (blood and PBS) and negative (Cefazolin) controls and assessed at five time points (0, 1, 4, 8 and 24 hours). RESULTS: After centrifugation, platelet count of PRPLP was 386 ± 65.5 × 103/?L and PRPHP was 867 ± 234.4 × 103/?L. Both PRP products showed a significant decrease (p<0.05) in bacterial growth at 8 hours compared to whole blood. CONCLUSION: The application of PRPLP and PRPHP showed a significant decrease in bacterial growth after 8 hours for S. aureus, S. epi, MRSA and P. acnes compared to the whole blood control group. S.epi, MRSA, and P. acnes also showed a significant decrease in bacterial growth after 24 hours. Despite differences in platelet concentration and WBC concentration, no difference in antibacterial activity was seen between the two preparations.
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PURPOSE: To assess molecular and histologic differences between the proximal (intra-articular) and distal (extra-articular) portions of the long head of the biceps (LHB) tendon in 3 different disease states (biceps instability, tendinosis, and degenerative joint disease [DJD]) compared with a healthy tendon (fresh frozen). METHODS: We used 32 LHB tendons of patients undergoing tenodesis (mean age, 54.7 ± 10.1 years) and 9 harvested tissue donors. Tendons were divided according to 4 diagnostic groups: (1) biceps instability, (2) tendinosis, (3) DJD, and (4) normal control. After sectioning, tendons were fixed in formalin and stained with H&E and alcian blue for histologic analysis. Measurements of collagen organization by use of polarized light microscopy was then performed, and protein expression for type I and type III collagen, tenascin C, and decorin was determined. RESULTS: There were no statistical differences found for protein expression of type I or type III collagen, tenascin C, or decorin. The proximal and distal regions of the tendons had statistically significant differences in alcian blue staining, with the proximal portion containing a higher amount of proteoglycan (instability, P = .001; tendinosis, P = .005; DJD, P = .008; control, P = .011). When compared with the nonpathologic control tendons, a significant increase in alcian blue staining for the proximal region was seen in all 3 groups. Total polarized light analysis showed that the distal tendon had a significantly higher intensity (organization) compared with the proximal tendon (P < .001); this was also seen in all of the diagnostic groups (instability, P = .010; tendinosis, P = .013; DJD, P = .07; control, P = .028). CONCLUSIONS: This study showed a greater degree of degeneration of the proximal (intra-articular) regions of the LHB tendon when compared with the distal regions in all pathologic groups. However, no major differences at the cellular level were found among groups. CLINICAL RELEVANCE: The pathomechanisms of the various forms of known LHB diagnoses are not yet fully understood and basic science studies may help in understanding their etiology and therefore optimizing treatment options.
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Artropatías/patología , Músculo Esquelético/patología , Articulación del Hombro/patología , Tendinopatía/patología , Tendones/patología , Adulto , Colágeno Tipo I/metabolismo , Colágeno Tipo III/metabolismo , Decorina/metabolismo , Femenino , Humanos , Artropatías/metabolismo , Masculino , Persona de Mediana Edad , Músculo Esquelético/metabolismo , Músculo Esquelético/cirugía , Osteoartritis/metabolismo , Osteoartritis/patología , Articulación del Hombro/anatomía & histología , Tenascina/metabolismo , Tendinopatía/metabolismo , Tendones/anatomía & histología , Tendones/metabolismo , Adulto JovenRESUMEN
PURPOSE: The purpose of this study was to quantify the extent of the anti-inflammatory effect of platelet-rich plasma (PRP) in a controlled in vitro environment. METHODS: Through the stimulation of human umbilical vein endothelial cells with inflammatory cytokines (tumor necrosis factor α and interferon γ), cell adhesion molecule expression (E-selectin, vascular cell adhesion molecule, and human leukocyte antigen DR) and PRP's anti-inflammatory effect can be measured. PRP was produced from 3 individuals using a single-spin (PRPLP) process. Treatment groups include negative (unstimulated) controls, positive (stimulated) controls, ketorolac tromethamine, methylprednisolone, PRP, ketorolac-PRP, and methylprednisolone-PRP. A fluorescence assay of the cellular inflammation markers was measured by the BioTek Synergy HT plate reader (BioTek Instruments, Winooski, VT) at 0, 1, 2, and 5 days. RESULTS: At days 2 and 5, methylprednisolone treatment showed a 2.1- to 5.8-fold reduction (P < .05) in inflammation markers over PRP. In addition, PRP and ketorolac showed a 1.4- to 2.5-fold reduction (P < .05) in cellular inflammation markers over the control. There was no statistically significant difference between ketorolac and PRP. CONCLUSIONS: Although PRP and ketorolac reduced cellular inflammation markers (E-selectin, vascular cell adhesion molecule, and human leukocyte antigen DR) compared with control, neither caused as great a reduction as methylprednisolone. CLINICAL RELEVANCE: Although PRP and ketorolac did not produce as significant a reduction in cellular inflammation markers as methylprednisolone, they reduced cellular inflammation compared with the control. These agents may have clinical application as injectable anti-inflammatory medications.
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Antiinflamatorios/farmacología , Células Endoteliales de la Vena Umbilical Humana/efectos de los fármacos , Ketorolaco/farmacología , Metilprednisolona/farmacología , Plasma Rico en Plaquetas/inmunología , Adulto , Células Cultivadas , Células Endoteliales de la Vena Umbilical Humana/inmunología , Humanos , Inflamación , Masculino , Adulto JovenRESUMEN
PURPOSE: The aim of this study was to examine the relations between age, gender, and number of viable mesenchymal stem cells (MSCs) in concentrated bone marrow (BM) obtained from the proximal humerus and distal femur during arthroscopic surgery. METHODS: BM was aspirated from either the proximal humerus (n = 55) or distal femur (n = 29) during arthroscopic surgery in 84 patients (51.3 ± 11.6 years). MSCs were obtained from fractionated bone marrow after a 5-minute spin at 1,500 rpm. Volume of BM and number of nucleated cells (NCs) were calculated, and samples were cultured for 6 days, after which point colony-forming units (CFUs) were quantified and fluorescence-activated cell sorting (FACS) analysis was performed. Simple linear regression was used to explore relations between age, gender, volume of aspirated BM, and MSCs per milliliter. RESULTS: BM aspirations yielded a mean quantity of 22.6 ± 12.3 mL. After centrifugation, 30.0 ± 16.7 × 10(6) nucleated cells/mL of concentrated BM were harvested. The proximal humerus provided 38.7 ± 52.6 × 10(6), and the distal femur, 25.9 ± 14.3 × 10(6), for an overall 766.3 ± 545.3 MSCs/mL of concentrated BM (proximal humerus: 883.9 ± 577.6, distal femur: 551.3 ± 408.1). Values did not significantly differ by age, gender, or donor site. CONCLUSIONS: Arthroscopic aspiration of bone marrow from the proximal humerus and distal femur is a reproducible technique and yields reliable concentrations of MSCs. The use of an intraoperative concentration method resulted in consistent amounts of MSCs in all clinically relevant age groups without a significant drop of the number of isolated MSCs. CLINICAL RELEVANCE: Human MSCs derived from concentrated bone marrow aspirate are a promising biological addition that may have practical use in the future of soft tissue augmentation. Arthroscopic techniques for bone marrow aspiration that do not require an additional surgical site for aspiration (e.g., iliac crest) or a second operative procedure may facilitate future use of MSCs in arthroscopic surgery.