A Systemic and Local Comparison of Senescence in an Acute Anterior Cruciate Ligament Injury-A Pilot Case Series.

BACKGROUND: Senescence, a characteristic of cellular aging and inflammation, has been linked to the acceleration of osteoarthritis. The purpose of this study is to prospectively identify, measure, and compare senescent profiles in synovial fluid and peripheral blood in patients with an acute knee injury within 48 h. METHODS: Seven subjects, aged 18-60 years, with an acute ACL tear with effusion were prospectively enrolled. Synovial fluid and peripheral blood samples were collected and analyzed by flow cytometry, using senescent markers C12FDG and CD87. The senescent versus pro-regenerative phenotype was probed at a gene and protein level using qRT-PCR and multiplex immunoassays. RESULTS: C12FDG and CD87 positive senescent cells were detected in the synovial fluid and peripheral blood of all patients. Pro-inflammatory IL-1ß gene expression measured in synovial fluid was significantly higher (p = 0.0156) than systemic/blood expression. Senescent-associated factor MMP-3 and regenerative factor TIMP-2 were significantly higher in synovial fluid compared to blood serum. Senescent-associated factor MMP-9 and regenerative factor TGFß-2 were significantly elevated in serum compared to synovial fluid. Correlation analysis revealed that C12FDG++/CD87++ senescent cells in synovial fluid positively correlated with age-related growth-regulated-oncogene (ρ = 1.00, p < 0.001), IFNγ (ρ = 1.00, p < 0.001), IL-8 (ρ = 0.90, p = 0.0374), and gene marker p16 (ρ = 0.83, p = 0.0416). CONCLUSIONS: There is an abundance of senescent cells locally and systemically after an acute ACL tear without a significant difference between those present in peripheral blood compared to synovial fluid. This preliminary data may have a role in identifying strategies to modify the acute environment within the synovial fluid, either at the time of acute ligament injury or reconstruction surgery.

Specific reprogramming of alpha cells to insulin-producing cells by short glucagon promoter-driven Pdx1 and MafA.

Endogenous reprogramming of pancreas-derived non-beta cells into insulin-producing cells is a promising approach to treat type 1 diabetes (T1D). One strategy that has yet to be explored is the specific delivery of insulin-producing essential genes, Pdx1 and MafA, to pancreatic alpha cells to reprogram the cells into insulin-producing cells in an adult pancreas. In this study, we used an alpha cell-specific glucagon (GCG) promoter to drive Pdx1 and MafA transcription factors to reprogram alpha cells to insulin-producing cells in chemically induced and autoimmune diabetic mice. Our results showed that a combination of a short glucagon-specific promoter with AAV serotype 8 (AAV8) can be used to successfully deliver Pdx1 and MafA to pancreatic alpha cells in the mouse pancreas. Pdx1 and MafA expression specifically in alpha cells were also able to correct hyperglycemia in both induced and autoimmune diabetic mice. With this technology, targeted gene specificity and reprogramming were accomplished with an alpha-specific promotor combined with an AAV-specific serotype and provide an initial basis to develop a novel therapy for the treatment of T1D.

Clinical Outcomes of Pectoralis Major Tendon Repair with and without Platelet-Rich Plasma.

Purpose: To assess clinical outcomes following pectoralis major tendon (PMT) repairs and to compare outcomes of PMT repairs augmented with and without leukocyte-poor platelet-rich plasma (LP-PRP). Methods: A retrospective review of prospectively collected data was performed of patients who underwent a PMT repair from May 2007 to June 2019 with a minimum of 2-year follow-up. Exclusion criteria included revision PMT repair, PMT reconstruction, and concomitant repair of another glenohumeral tendon/ligament. LP-PRP was injected surrounding the PMT repair before wound closure. Patient-reported outcome (PRO) data were collected preoperatively and evaluated at final follow-up using the American Shoulder and Elbow Surgeons Score (ASES), Single Assessment Numeric Evaluation Score (SANE), Quick Disabilities of the Arm, Shoulder and Hand Score (QuickDASH), and Short Form 12 physical component summary (SF-12 PCS), patient satisfaction with outcomes. Results: Twenty-three men (mean age, 38.6 years; range, 20.5-64.3 years) were included in the final analysis. Mean time from injury to surgery was 30 days (range, 3-123 days). Follow-up was obtained for 16 of 23 patients (70%) at a mean of 5.1 years (range 2.0-13.0 years). Significant improvement in PROs was observed (ASES: 59.0 → 92.4, P = .008; SANE: 44.4 → 85.9, P = .018; QuickDASH: 44.4 → 8.5, P = .018; and SF-12 PCS: 42.5 → 52.6, P = .008). Median satisfaction was 9 of 10 (range, 6-10). Patients receiving LP-PRP had superior ASES (99.6 vs 83.0, P = .001), SANE (94.8 vs 74.6, P = .005), QuickDASH (0.24 vs 19.1, P = .001), and patient satisfaction (10 vs 9, P = .037) scores compared with those without PRP. PROs were unchanged based on chronicity, mechanism of injury, or tear location. One patient had revision surgery at 3.4 years due to adhesions. Conclusions: PMT repair produces improved PROs at final follow-up when compared with preoperative values. Level of Evidence: Level III, retrospective comparative therapeutic trial.

The role of the aging microenvironment on the fate of PDGFRß lineage cells in skeletal muscle repair.

BACKGROUND: During aging, perturbation of muscle progenitor cell (MPC) constituents leads to progressive loss of muscle mass and accumulation of adipose and fibrotic tissue. Mesenchymal stem cells (MSCs) give rise to adipocytes and fibroblasts that accumulate in injured and pathological skeletal muscle through constitutive activation of platelet-derived growth factor receptors (PDGFRs). Although the role of the PDGFRα has been widely explored, there is a paucity of evidence demonstrating the role of PDGFRß in aged skeletal muscle. METHODS: In this study, we investigated the role of PDGFRß lineage cells in skeletal muscle during aging by using Cre/loxP lineage tracing technology. The PDGFR-Cre mice were crossed with global double-fluorescent Cre reporter mice (mTmG) that indelibly marks PDGFRß lineage cells. Those cells were analyzed and compared at different ages in the skeletal muscle of the mice. RESULTS: Our results demonstrated that PDGFRß lineage cells isolated from the muscles of young mice are MPC-like cells that exhibited satellite cell morphology, expressed Pax7, and undergo myogenic differentiation producing myosin heavy chain expressing myotubes. Conversely, the PDGFRß lineage cells isolated from muscles of old mice displayed MSC morphology with a reduced myogenic differentiation potential while expressing adipogenic and fibrotic differentiation markers. PDGFRß lineage cells also gave rise to newly regenerated muscle fibers in young mice after muscle injury, but their muscle regenerative process is reduced in old mice. CONCLUSIONS: Our data suggest that PDGFRß lineage cells function as MPCs in young mice, while the same PDGFRß lineage cells from old mice undergo a fate switch participating in adipose and fibrotic tissue infiltration in aged muscle. The inhibition of fate-switching in PDGFRß lineage cells may represent a potential approach to prevent fibrosis and fatty infiltration in skeletal muscle during the aging process.

Systematic Review of Platelet-Rich Plasma for Rotator Cuff Repair: Are We Adhering to the Minimum Information for Studies Evaluating Biologics in Orthopaedics?

BACKGROUND: The therapeutic efficacy of orthobiologic therapies for rotator cuff repair is difficult to evaluate owing to reporting inconsistences. In response, the Minimum Information for Studies Evaluating Biologics in Orthopaedics (MIBO) guidelines were developed to ensure standard reporting on orthobiologic therapies. PURPOSE: To systematically review clinical studies evaluating platelet-rich plasma (PRP) for full-thickness rotator cuff repair and adherence to MIBO guidelines. STUDY DESIGN: Scoping review; Level of evidence, 4. METHODS: A search was performed according to the PRISMA (Preferred Reporting Items for Systematic Reviews and Meta-Analyses) guidelines using PubMed, EMBASE, and the Cochrane Library databases. Inclusion criteria were clinical studies reporting on rotator cuff tears (≥1 cm) surgically repaired with PRP. Patient demographics, biologic intervention, and adherence to the MIBO guidelines were systematically reviewed. RESULTS: A total of 19 studies (1005 patients) were included in this review. Across all studies, 58.5% of the MIBO checklist items for PRP were reported. Out of 47 checklist items, 19 were reported in over 85% of studies, whereas 22 were reported in less than half of studies. Details of whole-blood processing and characteristics, as well as PRP processing and characteristics, were reported inconsistently, and no study provided adequate information to enable the precise replication of preparation protocols for PRP. CONCLUSION: This systematic review highlights the current reporting deficiencies within the scientific literature of important variables for evaluating PRP for full-thickness rotator cuff repair. There was widespread variability among published studies that evaluate PRP for this application and, more specifically, studies were limited by inconsistent universal reporting of whole-blood and PRP processing and postprocessing characteristics. To improve our understanding of biologic efficacy and to promote repeatability, stricter adherence to the MIBO guidelines is necessary. We propose that the checklist limitations be addressed and that modification of the MIBO guidelines be considered to improve the reporting of individual components within certain categories.

The Effect of a Single Freeze-Thaw Cycle on Matrix Metalloproteinases in Different Human Platelet-Rich Plasma Formulations.

Storing platelet-rich plasma (PRP) for future use is a compelling approach, presuming the retention of biological properties is maintained. However, certain factors in PRP preparations have deleterious effects for the treatment of certain musculoskeletal conditions. The purpose of this study was to measure and compare matrix metalloproteinase protein (MMP) concentrations between fresh and freeze-thawed leukocyte-rich PRP (LR-PRP) inactivated (LR-I) and activated (LR-A) preparations, and leukocyte-poor PRP (LP-PRP) inactivated (LP-I) and activated (LP-A) preparations. A volume of 60 mL of whole blood was drawn from 19 healthy donors. LP-I and LR-I samples were processed using a manual extraction and centrifugation methodology. LP-A and LR-A products were activated with 10% CaCl2 and recombinant thrombin. Blood fractions were either immediately assayed and analyzed or stored at -80 °C for 24, 72 and 160 h. Multiplex immunoassay was used to measure MMP-1, MMP-2, MMP-3, MMP-9, MMP-10, and MMP-12. MMP-1 concentrations increased in LR-A (p < 0.05) and MMP-9 significantly increased in LR-I (p < 0.05), while MMP-2 significantly decreased in LR-I (p < 0.05) and MMP-3 concentrations significantly decreased in LR-A (p < 0.05). MMP-12 concentrations also significantly decreased in LR-I (p < 0.05) from baseline concentrations. There were no significant differences between LP-A and LP-I preparations and MMP concentrations. MMP-10 concentrations in all PRP samples compared to each freezing time point were also not significantly different. MMPs regulate components of the extracellular matrix (ECM) in the remodeling phase of musculoskeletal injury. In this study, we observed a significant increase and decrease in MMP concentrations in response to a single freeze-thaw cycle in inactivated PRP and activated PRP preparations. This evidence contributes to the growing body of literature on the optimization of PRP preparation and storage strategies prior to delivery. Our findings suggest that specific PRP preparations after a single freeze-thaw may be more advantageous for certain musculoskeletal applications based on the presence of MMP concentrations.

Bone Marrow Concentrate Injection Treatment Improves Short-term Outcomes in Symptomatic Hip Osteoarthritis Patients: A Pilot Study.

BACKGROUND: Osteoarthritis (OA) is one of the leading causes of disability in the United States, the hip being the second most affected weightbearing joint. Autologous bone marrow concentrate (BMC) is a promising alternative therapy to conventional treatments, with the potential to mitigate inflammation and improve joint function. PURPOSE: To investigate the effectiveness of a single intra-articular BMC injection for patients with symptomatic hip OA. STUDY DESIGN: Case series; Level of evidence, 4. METHODS: A total of 24 patients diagnosed with symptomatic hip OA who elected to undergo a single BMC injection were prospectively enrolled in the study. Patients were excluded if they reported a preinjection Numeric Rating Scale (NRS) score for pain with activity of <6 points out of 10. The Western Ontario and McMaster Universities Arthritis Index (WOMAC), modified Harris Hip Score (mHHS), Hip Outcome Score-Activities of Daily Living (HOS-ADL), 12-Item Short Form Health Survey (SF-12), and NRS pain scores were collected before and after the procedure (6 weeks, 3 months, and 6 months). Joint space and Tönnis OA grade scores were recorded on preinjection anteroposterior pelvis radiographs. RESULTS: A total of 18 hips from 16 patients (7 male and 9 female) (mean age, 57.6 ± 11; mean body mass index, 25.9 ± 3.6 kg/m2) were used in the final analysis. Significant improvements were observed in NRS pain with activity (from 8 to 4.5; P < .001) and without activity (from 5 to 1; P < .001), WOMAC (from 31 to 16; P = .006), mHHS (from 63 to 80; P = .004), and HOS-ADL (from 71 to 85; P = .014) over 6 months. At 6 months, all patients maintained their improvements and did not return to preprocedure status. BMI significantly correlated with baseline WOMAC scores (P = .012) and inversely correlated with 6-month SF-12 Physical Component Summary (P = .038). Tönnis grades 2 and 3 were inversely correlated with 6-week SF-12 Mental Component Summary (P = .008) and 3-month pain with activity (P = .032). No serious adverse events were reported from the BMC harvest or injection procedure. CONCLUSION: A single BMC injection can significantly improve subjective pain and function scores up to 6 months in patients with symptomatic hip OA. Further studies are warranted to evaluate BMC treatment against other therapeutics in a larger sample size and compare the biological signature profiles that may be responsible for the therapeutic effect.

Effect of Oral Losartan on Orthobiologics: Implications for Platelet-Rich Plasma and Bone Marrow Concentrate-A Rabbit Study.

Recent efforts have focused on customizing orthobiologics, such as platelet-rich plasma (PRP) and bone marrow concentrate (BMC), to improve tissue repair. We hypothesized that oral losartan (a TGF-ß1 blocker with anti-fibrotic properties) could decrease TGF-ß1 levels in leukocyte-poor PRP (LP-PRP) and fibrocytes in BMC. Ten rabbits were randomized into two groups (N = 5/group): osteochondral defect + microfracture (control, group 1) and osteochondral defect + microfracture + losartan (losartan, group 2). For group 2, a dose of 10mg/kg/day of losartan was administrated orally for 12 weeks post-operatively. After 12 weeks, whole blood (WB) and bone marrow aspirate (BMA) samples were collected to process LP-PRP and BMC. TGF-ß1 concentrations were measured in WB and LP-PRP with multiplex immunoassay. BMC cell populations were analyzed by flow cytometry with CD31, CD44, CD45, CD34, CD146 and CD90 antibodies. There was no significant difference in TGF-ß1 levels between the losartan and control group in WB or LP-PRP. In BMC, the percentage of CD31+ cells (endothelial cells) in the losartan group was significantly higher than the control group (p = 0.008), while the percentage of CD45+ cells (hematopoietic cells-fibrocytes) in the losartan group was significantly lower than the control group (p = 0.03).

Heterogenetic parabiosis between healthy and dystrophic mice improve the histopathology in muscular dystrophy.

Duchenne muscular dystrophy (DMD) is a progressive muscle disease, characterized by mutations in the X-linked dystrophin, that has several therapeutic options but no curative treatment. Transplantation of muscle progenitor cells for treatment of DMD has been widely investigated; however, its application is hindered by limited cell survival due to the harmful dystrophic microenvironment. An alternative approach to utilize progenitor cells and circulatory factors and to improve the dystrophic muscle pathology and microenvironment is through parabiotic pairing, where mice are surgically sutured to create a joint circulatory system. Parabiotic mice were generated by surgically joining wild type (WT) mice expressing green fluorescent protein (GFP) with mdx mice. These mice developed a common circulation (approximately 50% green cells in the blood of mdx mice) 2-weeks after parabiotic pairing. We observed significantly improved dystrophic muscle pathology, including decreased inflammation, necrotic fibers and fibrosis in heterogenetic parabionts. Importantly, the GFP + cells isolated from the mdx mice (paired with GFP mice) underwent myogenic differentiation in vitro and expressed markers of mesenchymal stem cells and macrophages, which may potentially be involved in the improvement of dystrophic muscle pathology. These observations suggest that changing the dystrophic microenvironment can be a new approach to treat DMD.

Characterization of Growth Factors, Cytokines, and Chemokines in Bone Marrow Concentrate and Platelet-Rich Plasma: A Prospective Analysis.

BACKGROUND: Platelet-rich plasma (PRP) and bone marrow concentrate (BMC) are orthobiologic therapies with numerous growth factors and other bioactive molecules. Before the clinical utility of PRP and BMC is optimized as a combined therapy or monotherapy, an improved understanding of the components and respective concentrations is necessary. PURPOSE: To prospectively measure and compare anabolic, anti-inflammatory, and proinflammatory growth factors, cytokines, and chemokines in bone marrow aspirate (BMA), BMC, whole blood, leukocyte-poor PRP (LP-PRP), and leukocyte-rich PRP (LR-PRP) from samples collected and processed concurrently on the same day from patients presenting for elective knee surgery. STUDY DESIGN: Cross-sectional study; Level of evidence, 3. METHODS: Patients presenting for elective knee surgery were prospectively enrolled over a 3-week period. Whole blood from peripheral venous draw and BMA from the posterior iliac crest were immediately processed via centrifugation and manual extraction methods to prepare LR-PRP, LP-PRP, and BMC samples, respectively. BMA, BMC, whole blood, LR-PRP, and LP-PRP samples were immediately assayed and analyzed to measure protein concentrations. RESULTS: BMC had a significantly higher interleukin 1 receptor antagonist (IL-1Ra) concentration than all other preparations (all P < .0009). LR-PRP also had a significantly higher IL-1Ra concentration than LP-PRP (P = .0006). There were no significant differences in IL-1Ra concentration based on age, sex, body mass index, or chronicity of injury in all preparations. LR-PRP had significantly higher concentrations of platelet-derived growth factor AA (PDGF-AA) and PDGF-AB/BB than all other preparations (all P < .0006). LR-PRP also had significantly higher concentrations of matrix metalloproteinase 1 (MMP-1) and soluble CD40 ligand than all other preparations (all P < .004). LP-PRP had significantly higher concentrations of MMPs, namely MMP-2, MMP-3, and MMP-12, than all other preparations (all P < .007). CONCLUSION: BMC is a clinically relevant source of anti-inflammatory biologic therapy that may be more effective in treating osteoarthritis and for use as an intra-articular biologic source for augmented healing in the postsurgical inflammatory and healing phases, owing to its significantly higher concentration of IL-1Ra as compared with LR-PRP and LP-PRP. Additionally, LR-PRP had a significantly higher concentration of IL-1Ra than LP-PRP. In cases where increased vascularity and healing are desired for pathological or injured tissues, including muscle and tendon, LR-PRP may be optimal given its higher overall concentrations of PDGF, TGF-ß, EGF, VEGF, and soluble CD40 ligand.

The Influence of Naproxen on Biological Factors in Leukocyte-Rich Platelet-Rich Plasma: A Prospective Comparative Study.

PURPOSE: To quantify and compare normative catabolic and anabolic factor concentrations in leukocyte-rich platelet-rich plasma (LR-PRP) at various time points, including baseline, 1 week after initiating naproxen use, and after a 1-week washout period. METHODS: Asymptomatic healthy donors aged between 18 and 70 years were recruited (average age, 36.6 years; range, 25-64 years). Subjects were excluded from the study if they were actively taking any prescribed medications or nonsteroidal anti-inflammatory drugs (NSAIDs) or if they had any of the following at present or previously: blood or immunosuppression disorders, cancer, osteonecrosis, rheumatoid arthritis, avascular necrosis, NSAID intolerance, gastrointestinal or peptic ulcer disease, or kidney dysfunction. The anabolic factors vascular endothelial growth factor, fibroblast growth factor 2, platelet-derived growth factor AB (PDGF-AB), and platelet-derived growth factor AA (PDGF-AA) and the catabolic factors interleukin (IL) 1ß, IL-6, IL-8, and tumor necrosis factor α in LR-PRP were measured. Peripheral blood was drawn at 3 time points: baseline, after 1 week of naproxen use, and after a 1-week washout period. RESULTS: The angiogenic factors PDGF-AA (44% decrease in median) and PDGF-AB (47% decrease) significantly declined from baseline (P < .05) after 1 week of naproxen use. There was a significant recovery (P < .05) of PDGF-AA (94% increase) and PDGF-AB (153% increase) levels after the 1-week washout period, with a return to baseline levels. The catabolic factor IL-6 also had a significant decline from baseline (77% decrease in median, P < .05) after 1 week of naproxen use. After a 1-week washout period, the IL-6 level was similar to the baseline level (130% increase, P < .05). CONCLUSIONS: Naproxen use diminished several biological factors in LR-PRP; however, a 1-week washout period was sufficient for the recovery of PDGF-AA, PDGF-AB, and IL-6 to return to baseline levels. Tumor necrosis factor α, IL-1ß, IL-8, vascular endothelial growth factor, and fibroblast growth factor 2 did not show differences between the 3 time points of data collection. Discontinuing NSAIDs for a minimum of 1 week before LR-PRP treatment may improve certain biological factor levels. LEVEL OF EVIDENCE: Level II, prospective comparative study.

Current perspectives on biological approaches for osteoarthritis.

Musculoskeletal injuries that disrupt the structure and function of diarthrodial joints can cause permanent biomechanical alterations and lead to a more severe, chronic condition. Despite advancements that have been made to restore tissue function and delay the need for joint replacement, there are currently no disease-modifying therapies for osteoarthritis (OA). To reduce the risk of OA, innovative preventive medicine approaches have been developed over the last decade to treat the underlying pathology. Several biological approaches are promising treatment modalities for various stages of OA owing to their minimally invasive nature and actively dynamic physiological mechanisms that attenuate tissue degradation and inflammatory responses. Individualized growth factor and cytokine therapies, tissue-engineered biomaterials, and cell-based therapies have revolutionary potential for orthopedic applications; however, the paucity of standardization and categorization of biological components and their counterparts has made it difficult to determine their clinical and biological efficacy. Cell-based therapies and tissue-engineered biologics have become lucrative in sports medicine and orthopedics; nonetheless, there is a continued effort to produce a biological treatment modality tailored to target intra-articular structures that recapitulates tissue function. Advanced development of these biological treatment modalities will potentially optimize tissue healing, regeneration, and joint preservation strategies. Therefore, the purpose of this paper is to review current concepts on several biological treatment approaches for OA.

Platelet-Rich Plasma Augmentation for Hip Arthroscopy.

Biological augmentation and therapeutics are being increasingly used in musculoskeletal and orthopaedic care. Platelet-rich plasma (PRP) is produced from centrifugation of peripheral blood, a process that concentrates platelets within autologous plasma. The process of PRP preparation is fundamental in controlling the contents, and it influences its therapeutic potential. Platelets contain alpha granules that store and release a variety of growth factors and other proteins that may augment the healing environment; PRP also has the added benefit of promoting postsurgical hemostasis. The purpose of this report was to detail our institutional preparation protocol and method of administration of PRP during hip arthroscopy.