Identification and culture of meniscons, meniscus cells with their pericellular matrix.

BACKGROUND AIMS: Meniscus injury is highly debilitating and often results in osteoarthritis. Treatment is generally symptomatic; no regenerative treatments are available. "Chondrons," articular chondrocytes with preserved pericellular matrix, produce more hyaline cartilage extracellular matrix and improve cartilage repair. If meniscons exist in the meniscus and have similar therapeutic potential as chondrons, employing these cells has potential for meniscus cell therapy and tissue engineering. In this study, we isolated and cultured "meniscons," meniscus cells surrounded by their native pericellular matrix, and investigated cell behavior in culture compared with chondrons. METHODS: Human meniscons were enzymatically isolated from osteoarthritic menisci and cultured up to 28 days in fibrin glue. Freshly isolated meniscons and chondrons were analyzed by histology and transmission electron microscopy. We used 5-([4,6-dichlorotriazin-2-yl]amino)fluorescein hydrochloride labeling and type VI collagen immunohistochemistry to image pericellular matrix after 0 and 28 days of culture. Gene expression was quantified using real-time polymerase chain reaction and DNA content and proteoglycan production were analyzed using biochemical assays. RESULTS: Meniscons were successfully isolated from human meniscus tissue. The pericellular matrix of meniscons and chondrons was preserved during 28 days of culture. Meniscons and chondrons had similar cell proliferation and proteoglycan production. Meniscons and chondrons expressed similar levels of collagen type I alpha 1 chain, whereas collagen type II alpha 1 chain and aggrecan expression was lower in the meniscon population. CONCLUSIONS: Freshly isolated meniscons and meniscons cultured for 28 days share similarities with chondrons with regard to cell proliferation, morphology and biochemical activity. Rapid isolation of meniscons (45 min) demonstrates potential for one-stage meniscus regeneration and repair, which should be confirmed in vivo.

The use of melatonin for auditory brainstem response audiometry in children with comorbidities.

PURPOSE: In this study, the efficacy and feasibility of melatonin in young children with and without comorbidities, undergoing auditory brainstem response audiometry (ABR) was evaluated. The aim of this study was primarily to evaluate the use of melatonin for ABR investigations in children with comorbidities. Second, the efficacy of melatonin was evaluated based on several factors like sleep-onset latency, sleep duration, frequency of awakenings as well as adverse events. METHODS: Click-induced ABR tests were performed at the outpatient clinic between January, 2018 and August, 2020. Investigations were considered successful when binaural testing was completed. A dose of melatonin depending on age, 5 mg for children younger than 6 years and 10 mg if older than 6 years, was administered after placement of electrodes. RESULTS: 131 children were included in this study. 87% of all ABR investigations were performed successfully. Comorbidities such as neurodevelopmental disorders or developmental delays were present in 70% of all children. There was no significant difference in age (p = 0.36) or gender (p = 0.97) between the success and failed group. In addition, comorbidities were equally distributed between both groups. Mean sleep duration was 38 (SD 21) min and sleep-onset latency was 28 (SD 20) min No adverse events were documented. CONCLUSION: Melatonin is effective for ABR examinations in infants and children with and without comorbidities. Furthermore, it allows for sequential testing in those at risk for progressive hearing loss. Clear instructions to caregivers and expertise of audiologists are a prerequisite for optimal outcomes.

Fresh Versus Frozen Meniscal Allograft Transplant: Revisit or Redundant? A Systematic Review.

BACKGROUND: Fresh-frozen allografts are the current standard in meniscal allograft transplant (MAT) surgery, due to their availability, ease of preservation, and affordability. However, fresh-frozen grafts are associated with several clinical challenges such as graft shrinkage and extrusion, among many others. PURPOSE: To present the current knowledge on the use of fresh meniscal allografts, presenting whether benefits associated with fresh grafts provide sufficient evidence to support their use in clinical practice. STUDY DESIGN: Systematic review; Level of evidence, 5. METHODS: A comprehensive search was conducted with keywords listed below. After an initial screening on title and abstract, full-text articles were assessed with the inclusion criteria. RESULTS: A total of 78 studies matched the inclusion criteria. Literature and preclinical studies indicated that fresh meniscal allografts are beneficial for maintaining mechanical properties, graft ultrastructure, and matrix metabolism due to the presence of viable cells. Therefore, fresh allografts may address common complications associated with fresh-frozen MAT. To overcome challenges associated with both fresh-frozen and fresh allografts, a group has studied treating fresh-frozen allografts with a cell-based injection therapy. CONCLUSION: Fresh meniscal allografts pose several challenges including limited availability, demanding preservation procedures, and high costs. Although the role of viable cells within meniscal allografts remains controversial, these cells may be vital for maintaining tissue properties.

Cellular Enhancement of Frozen Meniscus Allograft Combining Native Meniscus and Mesenchymal Stromal Cell Injections.

OBJECTIVE: This proof-of-concept study investigated an improved cell-based injection therapy combining mesenchymal stem cells (MSCs) and meniscus cells (MCs) to support superior meniscus allograft repopulation and early revival compared to injecting MSCs alone. DESIGN: In this controlled laboratory study, frozen meniscus allograft samples were injected vertically with a cell suspension containing different ratios of MSCs and MCs or control (lactated ringers) and cultured for 28 days. Samples were analyzed weekly for cell viability, migration, and metabolism using histological and biochemical assays. Tissue medium was analyzed for matrix metalloproteinase (MMP) expression using zymography. RESULTS: Cellular repopulation of frozen allografts injected with different cell suspensions was validated by immunohistochemistry. Significant higher DNA content was evidenced in grafts treated with suspensions of MCs or MC:MSC (1:4 ratio). Cell metabolic activity was significantly different between all treated groups and control group after 1 week. Allografts injected with MCs showed significantly more cell proliferation than injections with MSCs. MMP2 activity was detected in medium of all grafts cellularized with MCs with or without MSCs. Scanning electron microscopy (SEM) analysis showed resolution of the needle puncture, but not in the control group. Cell labeling of MCs upon injection of mixed MC:MSC suspensions revealed a gradual increase in the cell ratio. CONCLUSIONS: The findings of this study establish that injection of MCs with or without MSCs enhances the cellularity of meniscus allograft to support early graft revival and remodeling.

Human meniscus allograft augmentation by allogeneic mesenchymal stromal/stem cell injections.

Meniscus allograft transplantations (MATs) represent established surgical procedures with proven outcomes. Yet, storage as frozen specimens and limited cellular repopulation may impair graft viability. This proof-of-concept study tests the feasibility of injecting allogeneic mesenchymal stromal/stem cells (MSCs) in meniscus allograft tissue. We investigated the injectable cell quantity, survival rate, migration, and proliferation ability of MSCs up to 28 days of incubation. In this controlled laboratory study, seven fresh-frozen human allografts were injected with human allogeneic MSCs. Cells were labeled and histological characteristics were microscopically imaged up to 28 days. Mock-injected menisci were included as negative controls in each experiment. Toluidine blue staining demonstrated that a 100-µl volume can be injected while retracting and rotating the inserted needle. Immediately after injection, labeled MSCs were distributed throughout the injection channel and eventually migrated into the surrounding tissues. Histological assessment revealed that MSCs cluster in disc-like shapes, parallel to the intrinsic lamination of the meniscus and around the vascular network. Quantification showed that more than 60% of cells were present in horizontally injected grafts and more than 30% were observed in vertically injected samples. On Day 14, cells adopted a spindle-shaped morphology and exhibited proliferative and migratory behaviors. On Day 28, live/dead ratio assessment revealed an approximately 80% cell survival. The study demonstrated the feasibility of injecting doses of MSCs (>0.1 million) in meniscus allograft tissue with active cell proliferation, migration, and robust cell survival.

Joint Surface Lesions in the Knee Treated with an Acellular Aragonite-Based Scaffold: A 3-Year Follow-Up Case Series.

OBJECTIVE: The study aimed to evaluate the clinical outcome and repair capacity of a cell-free aragonite-based scaffold in patients with an isolated symptomatic joint surface lesion (JSL) of the knee. DESIGN: Thirteen patients (age 33.5 ± 8.9; female 23%; body mass index 25.3 ± 3.4, K/L [Kellgren-Lawrence] 1.8) with a JSL (2.6 ± 1.7 cm2 [1.0-7.5 cm2]) of the distal femur were enrolled in a single-center prospective case series. Safety and clinical outcome was assessed by the KOOS (Knee Injury and Osteoarthritis Outcome Score), IKDC (International Knee Documentation Committee), Lysholm, and Tegner activity scale at baseline and 6, 12, 18, 24, and 36 months follow-up. The MOCART 2.0 and scaffold integration were evaluated on magnetic resonance imaging at 12, 24, and 36 months postoperatively. RESULTS: Primary outcome (KOOS pain) improved with 36.5 ± 14.7 points at 12 months (P = 0.002) and 41.2 ± 14.7 points at 36 months (P = 0.002) follow-up. Similar increasing trends were observed for the other KOOS subscales, IKDC, and Lysholm score, which were significantly better at each follow-up time point relative to baseline (P < 0.05). Activity level increased from 2.75 ± 1.6 to 4.6 ± 2.2 points at final follow-up (P = 0.07). The MOCART was 61.7 ± 12.6 at 12 months and 72.9 ± 13.0 at 36 months postoperatively. Sixty-six to 100% implant integration and remodeling was observed in 73.3% cases at 36 months. No serious adverse events were reported. CONCLUSION: The study demonstrated that the biphasic aragonite-based scaffold is a safe and clinically effective implant for treating small-medium sized JSLs of the distal femur in a young and active patient cohort. The implant showed satisfying osteointegration and restoration of the osteochondral unit up to 3 years postimplantation.