RESUMO
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.
RESUMO
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.
Assuntos
Lesões do Manguito Rotador , Humanos , Pessoa de Meia-Idade , Lesões do Manguito Rotador/tratamento farmacológico , Lesões do Manguito Rotador/cirurgia , Dasatinibe/farmacologia , Dasatinibe/uso terapêutico , Quercetina/farmacologia , Quercetina/uso terapêutico , Metaloproteinase 3 da Matriz/genética , Interleucina-6/metabolismo , Interleucina-8 , Senescência CelularRESUMO
Background: Connective tissue subacromial bursa-derived progenitor cells (SBDCs) have been suggested as a potent biologic augment to promote healing of the repaired rotator cuff tendon. Maximizing the amount of retained progenitor cells at the tendon repair site is essential for ensuring an optimal healing environment, warranting a search for proadhesive and proliferative adjuvants. The purpose was to evaluate the effect of magnesium (Mg), platelet-rich plasma (PRP), and a combination of both adjuvants on the in vitro cellular adhesion and proliferation potential of SBDCs on suture material commonly used in rotator cuff surgery. Methods: SBDCs were isolated from subacromial bursa samples harvested during rotator cuff repair and cultured in growth media. Commercially available collagen-coated nonabsorbable flat-braided suture was cut into 1-inch pieces, placed into 48-well culture dishes, and sterilized under ultraviolet light. Either a one-time dose of 5 mM sterile Mg, 0.2 mL of PRP, or a combination of both adjuvants was added, while a group without treatment served as a negative control. Cellular proliferation and adhesion assays on suture material were performed for each treatment condition. Results: Augmenting the suture with Mg resulted in a significantly increased cellular adhesion (total number of attached cells) of SBDCs compared to PRP alone (31,527 ± 19,884 vs. 13,619 ± 8808; P < .001), no treatment (31,527 ± 19,884 vs. 21,643 ± 8194; P = .016), and combination of both adjuvants (31,527 ± 19,884 vs. 17,121 ± 11,935; P < .001). Further, augmentation with Mg achieved a significant increase in cellular proliferation (absorbance) of SBDCs on suture material when compared to the PRP (0.516 ± 0.207 vs. 0.424 ± 0.131; P = .001) and no treatment (0.516 ± 0.207 vs. 0.383 ± 0.094; P < .001) group. The combination of Mg and PRP showed a significantly higher proliferation potential compared to PRP alone (0.512 ± 0.194 vs. 0.424 ± 0.131; P = .001) and no treatment (0.512 ± 0.194 vs. 0.383 ± 0.094; P < .001). There were no significant differences in the remaining intergroup comparisons (P > .05, respectively). Conclusion: Augmenting suture material with Mg resulted in a significantly increased cellular adhesion of SBDCs compared to untreated suture material, as well as augmentation with PRP alone or a combination of both adjuvants. Further, Mg with or without PRP augmentation achieved a significant increase in the cellular proliferation of SBDCs on suture material compared to untreated sutures and augmentation with PRP alone. Application of Mg may be a clinically feasible approach to optimizing the use of SBDCs as a biological augment in rotator cuff repair, while combined augmentation with PRP may harness the full potential for optimized tissue recovery due to the high concentration of PRP-derived growth factors.
RESUMO
BACKGROUND: It has been shown that subacromial bursa (SAB) harbors connective tissue progenitor cells. The purpose of this study was to evaluate the effects of implantation of SAB-derived cells (SBCs) suspended in a fibrin sealant bead and implantation of SAB tissue at rotator cuff repair site on biomechanical properties of the repair in a mouse (C57Bl/6) model of supraspinatus tendon (ST) detachment and repair. METHODS: Part 1: Murine SAB tissue was harvested and cultured. Viability of SBCs suspended in 10 µL of fibrin sealant beads was confirmed in vitro and in vivo. Eighty mice underwent right ST detachment and repair augmented with either fibrin sealant bead (control group) or fibrin sealant bead with 100,000 SBCs (study group) applied at the repair site. Part 2: 120 mice underwent right ST detachment and repair and were randomized equally into 4 groups: (1) a tissue group, which received a piece of freshly harvested SAB tissue; (2) a cell group, which received SBCs suspended in fibrin sealant bead; (3) a fibrin sealant group, which received plain fibrin sealant bead without cells; and (4) a control group, which received nothing at the ST repair site. An equal number of mice in each group were killed at 2 and 4 weeks. Specimens underwent biomechanical testing to evaluate failure force (part 1 and 2) and histologic analysis of the repair site (part 1 only). RESULTS: Part 1: The mean failure force in the study group was significantly higher than controls at 2 and 4 weeks (3.25 ± 1.03 N vs. 2.43 ± 0.56 N, P = .01, and 4.08 ± 0.99 N vs. 3.02 ± 0.8 N, P = .004, respectively). Mean cell density of the ST at the repair site was significantly lower in the study group at 2 weeks than in controls (18,292.13 ± 1706.41 vs. 29,501.90 ± 3627.49, P = .001). Study group specimens had lower proteoglycan contents than controls, but this difference was not statistically significant. Part 2: There was no difference in failure force between cell and tissue groups at the 2- and 4-week time points (P = .994 and P = .603, respectively). There was no difference in failure force between fibrin sealant bead and control groups at the 2- and 4-week time points (P = .978 and P = .752, respectively). CONCLUSION: This study shows that the application of SBCs and SAB tissue at the rotator cuff repair site increases the strength of repair in a murine model of rotator cuff detachment and repair.
Assuntos
Lesões do Manguito Rotador , Manguito Rotador , Camundongos , Animais , Manguito Rotador/cirurgia , Lesões do Manguito Rotador/cirurgia , Adesivo Tecidual de Fibrina/farmacologia , Adesivo Tecidual de Fibrina/uso terapêutico , Modelos Animais de Doenças , Camundongos Endogâmicos C57BL , ProteoglicanasRESUMO
PURPOSE: To build upon previous literature to identify a complete analysis of cellular contents of subacromial bursal tissue as well as the matrix surrounding the rotator cuff. METHODS: Samples of subacromial bursal tissue and surrounding matrix milieu from above the rotator cuff tendon and above the rotator cuff muscle bellies were obtained from 10 patients undergoing arthroscopic rotator cuff repair. Samples were analyzed using fluorescent-activated cell sorting and histologic analysis with staining protocols (Oil Red O, Alcian Blue, and Picro-Sirius Red), for identification of matrix components, including fat, proteoglycans, and collagen. RESULTS: Progenitor cells and fibroblast-type cells were present in significant amounts in subacromial bursal tissue in both tissues obtained from over the tendinous and muscle belly portions. Markers for neural tissue, myeloid cells, and megakaryocytes also were present to a lesser extent. There were prominent amounts of fat and proteoglycans present in the matrix, based on ImageJ analysis of stained histologic slides. CONCLUSIONS: The subacromial bursal tissue and surrounding matrix of patients undergoing rotator cuff repair contains progenitor cells in significant concentrations both over the tendon and muscle belly of the rotator cuff. CLINICAL RELEVANCE: This presence of progenitor cells, in particular, in the subacromial bursal tissue provides a potential basis for future applications of augmentation purposes in rotator cuff healing, and calls into question the practice of routine bursectomy. As the potential role of bursal tissue contents in growth and regeneration in the setting of rotator cuff healing is more well understood, maintaining this tissue may become more relevant. Concentration of these cellular components for use in autologous re-implantation is also an avenue of interest.
Assuntos
Lesões do Manguito Rotador , Manguito Rotador , Bolsa Sinovial/patologia , Bolsa Sinovial/cirurgia , Humanos , Manguito Rotador/cirurgia , Lesões do Manguito Rotador/patologia , Lesões do Manguito Rotador/cirurgia , Células-Tronco , Tendões/cirurgiaRESUMO
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.
Assuntos
Osteoblastos , Plasma Rico em Plaquetas , Adesão Celular , Proliferação de Células , Humanos , Ligamentos , Osteoblastos/fisiologia , SuturasRESUMO
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.
Assuntos
Células-Tronco Mesenquimais , Lesões do Manguito Rotador , Articulação do Ombro , Animais , Bolsa Sinovial/patologia , Diferenciação Celular , Camundongos , Manguito Rotador/patologia , Lesões do Manguito Rotador/patologiaRESUMO
BACKGROUND AND HYPOTHESIS: The treatment of periprosthetic joint infection is complicated by the presence of residual biofilm, which resists eradication owing to bacterial adherence to orthopedic implants. The purpose of this study was to compare Bactisure (Zimmer Biomet, Warsaw, IN, USA), povidone-iodine (Betadine), and chlorhexidine gluconate solution (Irrisept; Irrimax, Gainesville, FL, USA) in reducing biofilm formation of Staphylococcus aureus, Staphylococcus epidermidis, and Cutibacterium acnes inoculated on cobalt-chrome, titanium, and stainless steel disks, representing metals commonly used for shoulder arthroplasty. The hypothesis was that there would be no significant difference in biofilm reduction among the 3 topical adjuvants. METHODS: Strains of S aureus (ATCC 35556), S epidermidis (ATCC 35984), and C acnes (LMG 16711) were grown on cobalt-chrome, titanium, and stainless steel disks. For each strain, the disks were divided into 4 groups: (1) control, (2) povidone-iodine (Betadine), (3) chlorhexidine gluconate (Irrisept), and (4) Bactisure. Bacteria were grown on 5% sheep blood agar plates. Biofilm eradication was quantified using adenosine triphosphate bioluminescence and compared with controls 48 and 72 hours after implementation of the topical adjuvant. RESULTS: At 72 hours after implementation of the topical adjuvant, a statistically significant reduction in colony-forming units was observed for all topical adjuvants across all tested metals, as compared with their respective control. With respect to the topical adjuvants themselves, Bactisure more consistently demonstrated the most significant reduction in colony-forming units across all bacteria when the tested medium was adjusted for, with the exception of S aureus, which showed similar results to Betadine at 72 hours. CONCLUSION: By use of commonly encountered topical adjuvants on S aureus-, S epidermidis-, and C acnes-inoculated disks of various implant metals, a significant reduction in biofilm production was observed. Bactisure, a recent Food and Drug Administration-approved topical adjuvant, demonstrated the overall greatest efficacy of the agents studied.
Assuntos
Infecções Relacionadas à Prótese , Animais , Biofilmes , Próteses e Implantes , Infecções Relacionadas à Prótese/prevenção & controle , Ovinos , Staphylococcus aureus , Staphylococcus epidermidisRESUMO
PURPOSE: To evaluate whether nucleated cell count (NCC) could serve as an approximation for the number of colony-forming units (CFUs) in concentrated bone marrow aspirate (cBMA) obtained from the proximal humerus. METHODS: Bone marrow aspirate (BMA) was harvested from the proximal humerus in 96 patients (mean age 56.2 ± 7.0 years) during arthroscopic rotator cuff repair. Following concentration of the aspirate, nucleated cells of each sample were counted. The total number of CFUs was evaluated under the microscope at their first appearance, usually after 5 to 10 days in culture. Fluorescence-activated cell sorting analysis and assays for osteogenic, adipogenic, and chondrogenic differentiation were performed. Linear regression was assessed to predict the number of CFUs by using NCC. Age, sex, and body mass index (BMI) were evaluated as independent variables. RESULTS: The average volume of the obtained BMA was 86.7 ± 35.2 mL. The cBMA contained a mean of 26.3 ± 6.8 × 106 nucleated cells per mL, which yielded a mean of 1421.7 ± 802.7 CFUs in cell culture. There were no significant differences in NCC or number of CFUs when sex, volume of BMA, age, or BMI was examined independently (P >.05, respectively). Linear regression found that NCC was of limited predictive value for the total number of CFUs being yielded after cell culture (r2 = 0.28 with a root mean square error of 679.4). CONCLUSION: NCC was of negligible predictive value for the total number of CFUs for connective tissue progenitor cells in BMA harvested from the proximal humerus during arthroscopic rotator cuff repair. CLINICAL RELEVANCE: NCC is often used to assess the quality of cBMA samples for biological augmentation during surgery. The limited predictive value of this measurement tool is of clinical importance, because effectiveness of BMA applications has been suggested to depend on the concentration of progenitor cells within the sample.
Assuntos
Lesões do Manguito Rotador , Manguito Rotador , Artroscopia , Medula Óssea , Células da Medula Óssea , Contagem de Células , Tecido Conjuntivo , Humanos , Úmero , Pessoa de Meia-Idade , Manguito Rotador/cirurgia , Lesões do Manguito Rotador/cirurgia , Células-TroncoRESUMO
PURPOSE: To clinically evaluate patients who underwent a biologic augmentation technique in revision arthroscopic rotator cuff repair using an autologous fibrin scaffold and concentrated stem cells isolated from bone marrow aspirate (BMA) obtained from the proximal humerus. METHODS: This is a retrospective review of prospectively collected data from patients who underwent biologic augmentation of revision arthroscopic rotator cuff repair using an autologous fibrin scaffold and BMA obtained from the proximal humerus between 2014 and 2015. Minimum follow-up was 12 months. Outcome measures were collected preoperatively and postoperatively including range of motion as well as American Shoulder and Elbow Surgeons Shoulder Form, Simple Shoulder Test, single assessment numeric evaluation, and visual analog score. In addition, BMA samples of each patient were assessed for the number of nucleated cells and colony-forming units. Regression analysis was performed to investigate whether the number of nucleated cells and colony-forming units had an influence on outcome and failure. RESULTS: Ten patients who underwent biologic augmentation of revision arthroscopic rotator cuff repair using an autologous fibrin scaffold and concentrated BMA obtained from the proximal humerus between 2014 and 2015 were included. The mean follow-up time was 30.7 (range: 12-49) months. Four patients were revised at final follow-up. Postoperative clinical scores improved significantly: American Shoulder and Elbow Surgeons (28.1 ± 5.4 to 60.9 ± 9.0; P < .01), single assessment numeric evaluation (6.6 ± 2.3 to 65.1 ± 10.9; P < .01), visual analog scale (7.2 ± 0.9 to 3.1 ± 0.9; P < .01), and Simple Shoulder Test (1.6 ± 0.5 to 10.3 ± 5.7; P < .01). Postoperative range of motion increased significantly with regard to flexion (97.0 ± 13.6 to 151.0 ± 12.2; P < .01) and abduction (88.0 ± 14.0 to 134.0 ± 15.1; P = .038) but not with external rotation (38.0 ± 5.7 to 50.5 ± 6.5; P = .16). Less pain was correlated to an increased number of nucleated cells (P = .026); however, there was no correlation between failure rate and number of nucleated cells (P = .430). CONCLUSIONS: Patients who underwent biologic augmentation of revision arthroscopic rotator cuff repair using an autologous fibrin scaffold and concentrated BMA demonstrated a significant improvement in shoulder function along with reduction of pain. However, the overall revision rate for this procedure was 40%. LEVEL OF EVIDENCE: Level IV, therapeutic case series.
RESUMO
PURPOSE: To evaluate the effect of magnesium on cellular adhesion and proliferation of human subacromial bursal tissue (SBT), osteoblasts, and tenocytes on nonabsorbable suture tape commonly used in rotator cuff surgery. METHODS: Human SBT cells, primary human osteoblasts (HOBs), and primary human tenocytes were isolated from tissue samples and cultured in growth media. Commercially available collagen-coated nonabsorbable suture tape was cut into one-inch pieces, placed into 48-well culture dishes, sterilized under ultraviolet light, and treated with (+) or without (-) magnesium. For the (+) magnesium group, a one-time dose of 5 mM sterile magnesium chloride was added. Subsequently, cells were plated at a density of 20,000 cells/cm2. For each cell source (SBT, HOBs, tenocytes) cellular proliferation and adhesion assays on suture tape treated (+) or (-) magnesium were performed. RESULTS: SBT, HOBs, and tenocytes each demonstrated the ability to adhere and proliferate on suture tape. Augmenting suture tape with magnesium resulted in a significantly increased cellular adhesion of SBT compared with nonaugmented sutures (P = .001), whereas no significant differences were observed for HOBs (P = .081) and tenocytes (P = .907). Augmentation with magnesium demonstrated no significant difference in cellular proliferation of SBT (P = .856), HOBs (P = .672), and tenocytes (P = .251) compared with nonaugmented sutures. CONCLUSIONS: SBT, osteoblasts, and tenocytes each demonstrated the ability to adhere and proliferate on suture tape. In addition, augmenting the suture with magnesium resulted in a significantly increased cellular adhesion of SBT compared with nonaugmented sutures, whereas no significant differences were observed for osteoblasts and tenocytes. Further, magnesium did not impair the proliferative activity of SBT, osteoblasts, and tenocytes on suture tape used in rotator cuff surgery. CLINICAL RELEVANCE: Modifying the surface of the suture used for repair with application of magnesium may be an inexpensive and technically feasible option to improve the use of SBT for biologic augmentation of rotator cuff repair.
RESUMO
BACKGROUND: The role of subacromial bursa in rotator cuff pathology is unclear. Along with recognized inflammatory potential, current data demonstrate the presence of mesenchymal stem cells and potential regenerative properties of the bursa. The purpose of this study was to (1) approximate an in vitro co-culture model that represents interaction between torn rotator cuff tendon and subacromial bursa, (2) quantify the cellular activity of tendon and bursa and their interactions, (3) use this model to induce a state of inflammation present with rotator cuff pathology. METHODS: In part 1, tendon and bursa samples were obtained from 6 patients undergoing rotator cuff repair. Tendon and bursa were cultured alone and together in co-culture wells for 21 days. Markers specific for tenocyte gene expression (tenascin C, decorin, etc) were measured in both tendon and bursa alone and compared to co-culture models. In part 2 of the study, an inflammatory state was induced with interleukin-1ß treatment, and markers of inflammation were measured via protein assay at 0 and 21 days in samples from 7 additional patients. RESULTS: There was an increase in tendon and bursa markers in nearly all groups as evidenced by increased gene expression of known tendon and bursa markers. There was a significant increase in gene expression when torn tendon was co-cultured with bursa compared with culturing alone. Additionally, a state of inflammation was induced as evidenced by increased markers of inflammation, inflammatory protein concentration, and inflammatory cells and disruption of histologic morphology. CONCLUSION: There is a clear interaction between rotator cuff tendon and the milieu produced by the subacromial bursa in this in vitro co-culture system that is significantly different when compared to an isolated culture of tendon and bursa. This system was successfully used to induce a state of inflammation that may represent in vivo inflammation. This in vitro model of rotator cuff pathology can aid investigators in testing effects of agents proposed to improve rotator cuff healing. This can lead to further knowledge regarding effective treatment options.
Assuntos
Lesões do Manguito Rotador , Manguito Rotador , Bolsa Sinovial , Técnicas de Cocultura , Humanos , TendõesRESUMO
PURPOSE: To evaluate the time required for colonies to develop from concentrated bone marrow aspirate (cBMA) and subacromial bursal tissue samples. METHODS: Samples of cBMA and subacromial bursa tissue were harvested from patients undergoing rotator cuff repair surgery between November 2014 and December 2019. Samples were analyzed for time to form colonies and number of colonies formed. The impact of age, sex, and cellularity (cBMA only) was analyzed. Samples were cultured and evaluated daily for colony formation in accordance with the guidelines of the International Society for Cellular Therapy. Demographic factors were analyzed for impact on time to form colonies and number of colonies formed. RESULTS: Samples of cBMA were obtained from 92 patients. Subacromial bursa tissue was obtained from 54 patients. For cBMA, older age was associated with more days to form colonies (P = .003), but sex (P = .955) and cellularity (P = .623) were not. For bursa, increased age was associated with longer time to form colonies (P = .002) but not sex (P = .804). Conclusions: Increased age (in cBMA and subacromial bursa tissue) and lower initial cellularity (in cBMA) are associated with longer time to form colonies in culture. CLINICAL RELEVANCE: Although connective tissue progenitor cells are widely used in orthopaedic practice, there are few metrics to determine their efficacy. Time to form colonies may serve as an important measurement for determining connective tissue progenitor cell viability for augmentation of rotator cuff repair. Subacromial bursa tissue may represent a viable alternative to cBMA for augmentation of rotator cuff repair, capable of forming colonies expediently in vivo.
RESUMO
PURPOSE: To investigate the influence of patient demographics and rotator cuff tear characteristics on the cellular proliferation potential of subacromial bursa-derived cells (SBDCs). METHODS: Patients undergoing arthroscopic rotator cuff repair between December 2017 and February 2019 were considered for enrollment in the study. Basic demographic information as well as medical and surgical history were obtained for each patient. Subacromial bursa was harvested from over the rotator cuff tendon. Cellular proliferation was evaluated after 3 weeks of incubation by counting nucleated SBDCs. Fluorescence-activated cell sorting (FACS) analysis was performed to confirm the presence of mesenchymal stem cell (MSC) specific surface markers. Using preoperative radiographs and magnetic resonance imaging (MRI), acromiohumeral distance (AHD), severity of cuff tear arthropathy, and rotator cuff tear characteristics were evaluated. RESULTS: Seventy-three patients (mean age: 57.2 ± 8.5 years) were included in the study. There was no significant difference in cellular proliferation of SBDCs when evaluating the influence of age, sex, body mass index (BMI), smoking status, and presence of systemic comorbidities (p > .05, respectively). Similarly, there was no significant difference in cellular proliferation of SBDCs when looking at rotator cuff tear characteristics (size, tendon retraction, fatty infiltration, muscle atrophy), AHD, or severity of cuff tear arthropathy (p > .05). FACS analysis confirmed nucleated SBDCs to have a high positive rate of MSC specific surface markers. CONCLUSION: Subacromial bursa consistently demonstrated a high cellular proliferation potential regardless of patient demographics, rotator cuff tear characteristics, and severity of glenohumeral joint degeneration. CLINICAL RELEVANCE: These findings may alleviate concerns that subacromial bursa might lose cellular proliferation potential when being used for biologic augmentation in massive and degenerated rotator cuff tears, thus assisting in predicting tendon healing and facilitating surgical decision-making.
Assuntos
Artroscopia , Bolsa Sinovial/citologia , Lesões do Manguito Rotador/cirurgia , Manguito Rotador/cirurgia , Adulto , Idoso , Proliferação de Células , Células Cultivadas , Feminino , Humanos , Imageamento por Ressonância Magnética , Masculino , Células-Tronco Mesenquimais/citologia , Pessoa de Meia-Idade , Procedimentos de Cirurgia Plástica , Articulação do Ombro/cirurgia , Tendões/cirurgiaRESUMO
PURPOSE: The purpose of this study was to evaluate the number of colony-forming units (CFUs) derived from concentrated bone marrow aspirates (BMAs) that were processed following arthroscopic harvest from either the proximal humerus or the body of the ilium during biologic augmentation of the rotator cuff and acetabular labral repairs. METHODS: Between November 2014 and January 2019, BMA was harvested from the proximal humerus (n = 89) and the body of the ilium (n = 30) during arthroscopic surgery. Following concentration of the aspirate, a 0.5-mL aliquot was further processed and the number of nucleated cells (NC) was counted. Each aliquot was cultured until CFUs were quantifiable. Fluorescence-activated cell sorting analysis and quantitative polymerase chain reaction was performed to confirm presence of mesenchymal stem cells. BMA harvest sites were prospectively assessed and evaluated for differences in age, sex, volume of aspirated BM, and CFUs per milliliter of BMA. RESULTS: The prevalence (38.57 ± 27.92ilium vs. 56.00 ± 25.60humerus CFUs per 106 nucleated cells) and concentration (979.17 ± 740.31ilium vs. 1,516.62 ± 763.63humerus CFUs per 1.0 mL BMA) of CFUs was significantly higher (P < .001, respectively) for BMA harvested from the proximal humerus. Additionally, the estimated total number of cells was significantly higher (P = .013) in BMA from the proximal humerus (97,529.00 ± 91,064.01ilium vs. 130,552.4 ± 85,294.2humerus). There was no significant difference between groups regarding BMA volume (91.67 ± 18.77ilium vs. 85.63 ± 35.61humerus mL; P = .286) and NC count (24.01 ± 5.13ilium vs. 27.07 ± 6.28humerus × 106 per mL BMA; P = .061). The mean age was significantly lower (P < .001) in patients with BMA being harvested from the ilium (30.18 ± 7.63ilium vs. 56.82 ± 7.08humerus years). Patient sex and age had no significant influence on cellular measures within groups (P > .05, respectively). CONCLUSION: Both proximal humerus and the body of the ilium can be considered reliable sources of bone marrow aspirate for the use in biologic augmentation during their respective arthroscopic surgery. Samples of bone marrow aspirate from the proximal humerus yielded a significantly higher amount of CFUs when compared with samples of BMA obtained from the ilium. LEVEL OF EVIDENCE: Level II- prospective laboratorial study.
Assuntos
Artroscopia , Transplante de Medula Óssea , Medula Óssea , Úmero/patologia , Ílio/patologia , Manguito Rotador/cirurgia , Adipogenia , Adulto , Cartilagem Articular , Contagem de Células , Diferenciação Celular , Separação Celular , Condrogênese , Feminino , Citometria de Fluxo , Humanos , Masculino , Células-Tronco Mesenquimais/citologia , Pessoa de Meia-Idade , Osteogênese , Estudos Prospectivos , Células-Tronco , Adulto JovemRESUMO
PURPOSE: To classify subacromial bursal tissue using intraoperative and in vitro characteristics from specimens harvested during arthroscopic shoulder surgery. METHODS: Subacromial bursa was harvested over the rotator cuff from 48 patients (57 ± 10 years) undergoing arthroscopic shoulder surgery. Specimens were characterized intraoperatively by location (over rotator cuff tendon or muscle), tissue quality (percent of either fatty or fibrous infiltration), and vascularity before complete debridement. Nucleated cell counts were determined after 3 weeks incubation and histological sections were reviewed for degree of fatty infiltration and vascularity. Mesenchymal stem cell surface markers were counted via flow cytometry (n = 3) and cellular migration was observed using a fluoroscopic assay (n = 3). RESULTS: Intraoperatively, muscle bursa was found most often to have >50% fatty infiltration (n = 39), whereas tendon bursa showed majority fibrous tissue (n = 32). Cellular proliferation did not significantly differ according to intraoperative tissue quality. Intraoperative vascularity was associated with greater proliferation for highly vascular samples (P = 0.023). Tendon bursa demonstrated significantly greater proliferation potential than muscle bursa (P = 0.00015). Histologic assessment of fatty infiltration was moderately correlated with gross tissue fattiness (ρ = -0.626, P = 7.14 × 10-11). Flow cytometry showed that 90% to 100% of bursal cells were positive for MSC surface markers. Peak cellular migration rates occurred between 18 and 30 hours' incubation. CONCLUSIONS: Intraoperative and in vitro subacromial bursa characteristics were not found to reliably correlate with the degree of cellular proliferation. However, the anatomic location of subacromial bursa was consistently predictive of increased proliferation potential. Bursa-derived nucleated cells were confirmed to include mesenchymal stem cells with migratory potential. CLINICAL RELEVANCE: The anatomic distinction between muscle and tendon bursa provides a simple classification for predicting cellular activity.
Assuntos
Bolsa Sinovial/patologia , Artropatias/patologia , Artropatias/cirurgia , Articulação do Ombro/patologia , Articulação do Ombro/cirurgia , Adulto , Idoso , Artroscopia , Biópsia , Bolsa Sinovial/cirurgia , Proliferação de Células , Feminino , Humanos , Masculino , Células-Tronco Mesenquimais/patologia , Pessoa de Meia-Idade , Músculo Esquelético/patologia , Ombro/patologia , Ombro/cirurgia , Tendões/patologiaRESUMO
PURPOSE: To evaluate the clinical outcomes of patients who underwent biologically enhanced patch augmentation repair for the treatment of revision massive rotator cuff tears. METHODS: Twenty-two patients who underwent arthroscopic and mini-open rotator cuff repair using a patch augmented with platelet-rich plasma and concentrated bone marrow aspirate (cBMA) for revision massive (≥2 tendons) rotator cuff tears from 2009 to 2014, with a minimum 1-year follow-up, were included in the study. In this procedure the medial side of the graft is secured to the rotator cuff tendon remaining medially. American Shoulder and Elbow Surgeons (ASES), Simple Shoulder Test, and postoperative Single Assessment Numerical Evaluation scores were evaluated. To determine the clinical relevance of ASES scores, the minimal clinically important difference, substantial clinical benefit (SCB), and the patient-acceptable symptomatic state (PASS) thresholds were used. Clinical success or failure was defined based on whether the patient reached the SCB threshold. In the laboratory, cellular counting along with the concentration of connective tissue progenitor cells were performed on patch samples from the day of surgery. Scaffolds were processed histologically at days 0, 7,14, and 21 of culture. RESULTS: Patients had significant improvement in the Simple Shoulder Test (2.6 ± 3.0pre vs 5.2 ± 4.2post, P = .01), whereas improvement in pain scores was found to be nonsignificant (5.6 ± 2.5pre vs 4.2 ± 3.4post,P = .11) at final follow-up. Mean ASES improved by Δ14.6 ± 33.4 points; however, this did not reach statistical significance (40.2 ± 21.6pre vs 53.9 ± 31.4post,P = .10). With regards to ASES score, 45% of patients achieved the minimal clinically important difference, 41% the SCB, and 32% reached or exceeded the PASS criteria. At 21 days, there was a significantly greater cell count in scaffolds from patients who had clinical success than those who were failures (P = .02). CONCLUSIONS: Only 41% of patients undergoing biologically enhanced patch augmentation repair reached substantial clinical benefit, whereas 32% reached or exceeded the PASS criteria. LEVEL OF EVIDENCE: Case Series: Level IV.
Assuntos
Lesões do Manguito Rotador/cirurgia , Telas Cirúrgicas , Artroscopia , Medula Óssea , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Plasma Rico em Plaquetas , Complicações Pós-Operatórias , Amplitude de Movimento Articular , Lesões do Manguito Rotador/patologia , Resultado do TratamentoRESUMO
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.
Assuntos
Acetábulo/cirurgia , Artroscopia/métodos , Células da Medula Óssea/citologia , Doenças das Cartilagens/cirurgia , Cartilagem Articular/cirurgia , Células-Tronco/citologia , Coleta de Tecidos e Órgãos/métodos , Acetábulo/patologia , Adulto , Doenças das Cartilagens/patologia , Cartilagem Articular/patologia , Contagem de Células , Feminino , Seguimentos , Humanos , Ílio , Masculino , Pessoa de Meia-Idade , Estudos Prospectivos , Adulto JovemRESUMO
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.
Assuntos
Artroscopia/métodos , Bolsa Sinovial/cirurgia , Células-Tronco Mesenquimais/citologia , Procedimentos de Cirurgia Plástica/métodos , Lesões do Manguito Rotador/cirurgia , Manguito Rotador/cirurgia , Cicatrização , Contagem de Células , Feminino , Humanos , Masculino , Pessoa de Meia-IdadeRESUMO
BACKGROUND: Low aspiration volumes have been recommended to allow for higher concentrations of progenitor cells during bone marrow harvesting. However, these guidelines then require multiple aspiration attempts in order to maximize cellular yield. The purpose of this study was to investigate the effect of a single, high-volume aspiration with four consecutive aliquots on the number of nucleated cells (NCs) and colony-forming units (CFUs) in concentrated bone marrow aspirate (cBMA) taken from the proximal humerus. METHODS: cBMA was taken from the proximal humerus of patients undergoing arthroscopic rotator cuff surgery. Four 12-mL double syringes were used consecutively from a single trocar to obtain four 10 cc aliquots. Each then underwent centrifugation to create a fractionated layer rich in nucleated cells. Following cellular separation, NCs were counted and CFUs were evaluated after incubation of 7-10 days. Cellular comparisons between each aliquot were performed along with their interaction with patient age and sex. RESULTS: Twenty-nine patients (55.9 ± 4.6 years) were included in this study. The number of NCs and CFUs showed significant differences between the four aliquots of aspirate, with the first 10 cc aliquot providing the highest amount (p < 0.001, respectively). No significant differences were found between the sum of the three sequential aliquots compared to the initial 10 cc sample. There were no significant differences between male and female patients (p > 0.05). Increasing age resulted in no significant decrease in the number of NCs and CFUs across the four consecutive aliquots (p > 0.05). CONCLUSION: In conclusion, while the initial aliquot provided the greatest number of nucleated cells and cultured CFUs, the addition of each sequential volume aspirate yielded similar amounts in total. This demonstrates the potential effectiveness of obtaining of higher volume aspirates from the proximal humerus during rotator cuff repair.