Modernizing Storage Conditions for Fresh Osteochondral Allografts by Optimizing Viability at Physiologic Temperatures and Conditions

Cartilage ◽  
2019 ◽  
pp. 194760351988879
Author(s):  
Janet M. Denbeigh ◽  
Mario Hevesi ◽  
Carlo A. Paggi ◽  
Zachary T. Resch ◽  
Leila Bagheri ◽  
...  
Keyword(s):  
Gene Expression ◽  
Clinical Success ◽  
Quantitative Polymerase Chain Reaction ◽  
Viable Cell ◽  
Clinical Results ◽  
Storage Conditions ◽  
Viable Cell Density ◽  
Osteochondral Allograft ◽  
Cartilage Defects ◽  
Chondrocyte Viability

Objective. Osteochondral allograft (OCA) transplantation has demonstrated good long-term outcomes in treatment of cartilage defects. Viability, a key factor in clinical success, decreases with peri-implantation storage at 4°C during pathogen testing, matching logistics, and transportation. Modern, physiologic storage conditions may improve viability and enhance outcomes. Design. Osteochondral specimens from total knee arthroplasty patients (6 males, 5 females, age 56.4 ± 2.2 years) were stored in media and incubated at normoxia (21% O2) at 22°C or 37°C, and hypoxia (2% O2) at 37°C. Histology, live-dead staining, and quantitative polymerase chain reaction (qPCR) was performed 24 hours after harvest and following 7 days of incubation. Tissue architecture, cell viability, and gene expression were analyzed. Results. No significant viability or gene expression deterioration of cartilage was observed 1-week postincubation at 37°C, with or without hypoxia. Baseline viable cell density (VCD) was 94.0% ± 2.7% at day 1. At day 7, VCD was 95.1% (37°C) with normoxic storage and 92.2% (37°C) with hypoxic storage ( P ≥ 0.27). Day 7 VCD (22°C) incubation was significantly lower than both the baseline and 37°C storage values (65.6%; P < 0.01). COL1A1, COL1A2, and ACAN qPCR expression was unchanged from baseline ( P < 0.05) for all storage conditions at day 7, while CD163 expression, indicative of inflammatory macrophages and monocytes, was significantly lower in the 37°C groups ( P < 0.01). Conclusion. Physiologic storage at 37°C demonstrates improved chondrocyte viability and metabolism, and maintained collagen expression compared with storage at 22°C. These novel findings guide development of a method to optimize short-term fresh OCA storage, which may lead to improved clinical results.

scholarly journals Beneficial Storage Effects of Epigallocatechin-3-O-Gallate on the Articular Cartilage of Rabbit Osteochondral Allografts

2009 ◽  
Vol 18 (5-6) ◽  
pp. 505-512 ◽  
Author(s):  
Jung Yoon Bae ◽  
Kazuaki Matsumura ◽  
Shigeyuki Wakitani ◽  
Amu Kawaguchi ◽  
Sadami Tsutsumi ◽  
...  
Keyword(s):  
Articular Cartilage ◽  
Disease Transmission ◽  
Culture Medium ◽  
Clinical Success ◽  
Osteochondral Allograft ◽  
Cartilage Defects ◽  
Clinical Effects ◽  
Normal Cells ◽  
Osteochondral Grafts ◽  
Osteochondral Allografts

A fresh osteochondral allograft is one of the most effective treatments for cartilage defects of the knee. Despite the clinical success, fresh osteochondral allografts have great limitations in relation to the short storage time that cartilage tissues can be well-preserved. Fresh osteochondral grafts are generally stored in culture medium at 4°C. While the viability of articular cartilage stored in culture medium is significantly diminished within 1 week, appropriate serology testing to minimize the chances for the disease transmission requires a minimum of 2 weeks. (–)-Epigallocatechin-3- O-gallate (EGCG) has differential effects on the proliferation of cancer and normal cells, thus a cytotoxic effect on various cancer cells, but a cytopreservative effect on normal cells. Therefore, a storage solution containing EGCG might extend the storage duration of articular cartilages. Rabbit osteochondral allografts were performed with osteochondral grafts stored at 4°C in culture medium containing EGCG for 2 weeks and then the clinical effects were examined with macroscopic and histological assessment after 4 weeks. The cartilaginous structure of an osteochondral graft stored with EGCG was well-preserved with high cell viability and glycosaminoglycan (GAG) content of the extracellular matrix (ECM). After an osteochondral allograft, the implanted osteochondral grafts stored with EGCG also provided a significantly better retention of the articular cartilage with viability and metabolic activity. These data suggest that EGCG can be an effective storage agent that allows long-term preservation of articular cartilage under cold storage conditions.

Review of Mechanical, Processing, and Immunologic Factors Associated With Outcomes of Fresh Osteochondral Allograft Transplantation of the Talus

2017 ◽  
Vol 38 (7) ◽  
pp. 808-819 ◽  
Author(s):  
Chikezie N. Okeagu ◽  
Erin A. Baker ◽  
Nicholas A. Barreras ◽  
Zachary M. Vaupel ◽  
Paul T. Fortin ◽  
...  
Keyword(s):  
Clinical Outcomes ◽  
Storage Conditions ◽  
Lesion Size ◽  
Cartilage Tissue ◽  
Osteochondral Allograft ◽  
Osteochondral Lesions ◽  
Level Of Evidence ◽  
Chondrocyte Viability ◽  
Immunological Mechanisms ◽  
Graft Storage

Osteochondral lesions of the talus (OLTs) are an increasingly implicated cause of ankle pain and instability. Several treatment methods exist with varying clinical outcomes. Due in part to successful osteochondral allografting (OCA) in other joints, such as the knee and shoulder, OCA has gained popularity as a treatment option, especially in the setting of large lesions. The clinical outcomes of talar OCA have been inconsistent relative to the positive results observed in other joints. Current literature regarding OCA failure focuses mainly on 3 factors: the effect of graft storage conditions on chondrocyte viability, graft/lesion size, and operative technique. Several preclinical studies have demonstrated the ability for bone and cartilage tissue to invoke an immune response, and a limited number of clinical studies have suggested that this response may have the potential to influence outcomes after transplantation. Further research is warranted to investigate the role of immunological mechanisms as an etiology of OCA failure. Level of Evidence: Level V, expert opinion.

Fresh Osteochondral Allograft Transplantation in the Knee: A Viability and Histologic Analysis for Optimizing Graft Viability and Expanding Existing Standard Processed Graft Resources Using a Living Donor Cartilage Program

Cartilage ◽  
2019 ◽  
pp. 194760351988033
Author(s):  
Mario Hevesi ◽  
Janet M. Denbeigh ◽  
Carlo A. Paggi ◽  
Catalina Galeano-Garces ◽  
Leila Bagheri ◽  
...  
Keyword(s):  
Living Donor ◽  
Ex Vivo ◽  
Viable Cell ◽  
Viable Cell Density ◽  
Osteochondral Allograft ◽  
Clinical Grade ◽  
Allograft Transplantation ◽  
Graft Harvest ◽  
Healthy Cartilage ◽  
Total Knee

Objective This study aims to (1) determine and validate living cartilage allograft transplantation as a novel source for viable osteochondral allograft (OCA) tissues and (2) perform histologic and viability comparisons of living donor cartilage tissues to currently available clinical-grade standard processed grafts. Design Using healthy cartilage from well-preserved contralateral compartments in 27 patients undergoing total knee arthroplasty (TKA) and 10 clinical-grade OCA specimens obtained immediately following operative implantation, standard and living donor OCA quality was evaluated at the time of harvest and following up to 3 weeks of storage on the basis of macroscopic International Cartilage Repair Society grade, histology, and viability. Results Osteochondral samples demonstrated a consistent decrease in viability and histologic quality over the first 3 weeks of storage at 37°C, supporting the utility of an OCA paradigm shift toward early implantation, as was the clinical standard up until recent adoption of transplantation at 14 to 35 days following donor procurement. Samples from the 10 clinical-grade OCAs, implanted at an average of 23 days following graft harvest demonstrated a mean viable cell density of 45.6% at implantation, significantly lower ( P < 0.01) than the 93.6% viability observed in living donor allograft tissues. Conclusions Osteochondral tissue viability and histologic quality progressively decreases with ex vivo storage, even when kept at physiologic temperatures. Currently available clinical OCAs are stored for 2 to 5 weeks prior to implantation and demonstrate inferior viability to that of fresh osteochondral tissues that can be made available through the use of a living donor cartilage program.

Analysis of Stored Osteochondral Allografts at the Time of Surgical Implantation

2005 ◽  
Vol 33 (10) ◽  
pp. 1479-1484 ◽  
Author(s):  
R. Todd Allen ◽  
Catherine M. Robertson ◽  
Andrew T. Pennock ◽  
William D. Bugbee ◽  
Frederick L. Harwood ◽  
...  
Keyword(s):  
Articular Cartilage ◽  
Cell Viability ◽  
Cell Density ◽  
Tissue Bank ◽  
Viable Cell ◽  
Viable Cell Density ◽  
Proteoglycan Synthesis ◽  
Superficial Zone ◽  
Chondrocyte Viability ◽  
Osteochondral Allografts

Background To date, the morphological, biochemical, and biomechanical characteristics of articular cartilage in osteochondral allografts that have been stored have not been fully described. Hypothesis Osteochondral allografts procured and stored commercially for a standard period as determined by tissue banking protocol will have compromised chondrocyte viability but preserved extracellular matrix quality. Study Design Controlled laboratory study. Methods Unused cartilage from 16 consecutive osteochondral allografts was sampled during surgery after tissue bank processing and storage. Ten grafts were examined for cell viability and viable cell density using confocal microscopy, proteoglycan synthesis via 35SO4 uptake, and glycosaminoglycan content and compared with fresh cadaveric articular cartilage. Biomechanical assessment was performed on the 6 remaining grafts by measuring the indentation stiffness of the cartilage. Results The mean storage time for the transplanted specimens was 20.3 ± 2.9 days. Chondrocyte viability, viable cell density, and 35SO4 uptake were significantly lower in allografts at implantation when compared to fresh, unstored controls, whereas matrix characteristics, specifically glycosaminoglycan content and biomechanical measures, were unchanged. In addition, chondrocyte viability in the stored allografts was preferentially decreased in the superficial zone of cartilage. Conclusion Human osteochondral allografts stored for a standard period (approximately 3 weeks) before implantation undergo decreases in cell viability, especially in the critically important superficial zone, as well as in cell density and metabolic activity, whereas matrix and biomechanical characteristics appear conserved. The exact clinical significance of these findings, however, is unknown, as there are no prospective studies examining clinical outcomes using grafts stored for extended periods. Clinical Relevance Surgeons who perform this procedure should understand the cartilage characteristics of the graft after 21 days of commercial storage in serum-free media.

Anti-Inflammatory Effects of Novel Standardized Platelet Rich Plasma Releasates on Knee Osteoarthritic Chondrocytes and Cartilage in vitro

2019 ◽  
Vol 20 (11) ◽  
pp. 920-933 ◽  
Author(s):  
Lucía Gato-Calvo ◽  
Tamara Hermida-Gómez ◽  
Cristina R. Romero ◽  
Elena F. Burguera ◽  
Francisco J. Blanco
Keyword(s):  
Gene Expression ◽  
Ex Vivo ◽  
Platelet Rich Plasma ◽  
Cartilage Explants ◽  
Ex Vivo Model ◽  
Chondrocyte Viability ◽  
Platelet Concentration ◽  
The Absolute ◽  
Anti Inflammatory

Background: Platelet Rich Plasma (PRP) has recently emerged as a potential treatment for osteoarthritis (OA), but composition heterogeneity hampers comparison among studies, with the result that definite conclusions on its efficacy have not been reached. Objective: 1) To develop a novel methodology to prepare a series of standardized PRP releasates (PRP-Rs) with known absolute platelet concentrations, and 2) To evaluate the influence of this standardization parameter on the anti-inflammatory properties of these PRP-Rs in an in vitro and an ex vivo model of OA. Methods: A series of PRPs was prepared using the absolute platelet concentration as the standardization parameter. Doses of platelets ranged from 0% (platelet poor plasma, PPP) to 1.5·105 platelets/µl. PRPs were then activated with CaCl2 to obtain releasates (PRP-R). Chondrocytes were stimulated with 10% of each PRP-R in serum-free culture medium for 72 h to assess proliferation and viability. Cells were co-stimulated with interleukin (IL)-1β (5 ng/ml) and 10% of each PRP-R for 48 h to determine the effects on gene expression, secretion and intra-cellular content of common markers associated with inflammation, catabolism and oxidative stress in OA. OA cartilage explants were co-stimulated with IL-1β (5 ng/ml) and 10% of either PRP-R with 0.75·105 platelets/µl or PRP-R with 1.5·105 platelets/µl for 21 days to assess matrix inflammatory degradation. Results: Chondrocyte viability was not affected, and proliferation was dose-dependently increased. The gene expression of all pro-inflammatory mediators was significantly and dose-independently reduced, except for that of IL-1β and IL-8. Immunoblotting corroborated this effect for inducible NO synthase (NOS2). Secreted matrix metalloproteinase-13 (MMP-13) was reduced to almost basal levels by the PRP-R from PPP. Increasing platelet dosage led to progressive loss to this anti-catabolic ability. Safranin O and toluidine blue stains supported the beneficial effect of low platelet dosage on cartilage matrix preservation. Conclusion: We have developed a methodology to prepare PRP releasates using the absolute platelet concentration as the standardization parameter. Using this approach, the composition of the resulting PRP derived product is independent of the donor initial basal platelet count, thereby allowing the evaluation of its effects objectively and reproducibly. In our OA models, PRP-Rs showed antiinflammatory, anti-oxidant and anti-catabolic properties. Platelet enrichment could favor chondrocyte proliferation but is not necessary for the above effects and could even be counter-productive.

scholarly journals Mapping the molecular basis for growth related phenotypes in industrial producer CHO cell lines using differential proteomic analysis

2021 ◽  
Vol 21 (1) ◽  
Author(s):  
Laura Bryan ◽  
Michael Henry ◽  
Ronan M. Kelly ◽  
Christopher C. Frye ◽  
Matthew D. Osborne ◽  
...  
Keyword(s):  
Cell Culture ◽  
Cell Density ◽  
Cell Lines ◽  
Viable Cell ◽  
Viable Cell Density ◽  
High Peak ◽  
Proteomic Profiling ◽  
Cho Cell ◽  
Extended Culture ◽  
Key Pathways

Abstract Background The ability to achieve high peak viable cell density earlier in CHO cell culture and maintain an extended cell viability throughout the production process is highly desirable to increase recombinant protein yields, reduce host cell impurities for downstream processing and reduce the cost of goods. In this study we implemented label-free LC-MS/MS proteomic profiling of IgG4 producing CHO cell lines throughout the duration of the cell culture to identify differentially expressed (DE) proteins and intracellular pathways associated with the high peak viable cell density (VCD) and extended culture VCD phenotypes. Results We identified key pathways in DNA replication, mitotic cell cycle and evasion of p53 mediated apoptosis in high peak VCD clonally derived cell lines (CDCLs). ER to Golgi vesicle mediated transport was found to be highly expressed in extended culture VCD CDCLs while networks involving endocytosis and oxidative stress response were significantly downregulated. Conclusion This investigation highlights key pathways for targeted engineering to generate desirable CHO cell phenotypes for biotherapeutic production.

scholarly journals Modulation of the Tissue Expression Pattern of Zebrafish CRP-Like Molecules Suggests a Relevant Antiviral Role in Fish Skin

Biology ◽  
2021 ◽  
Vol 10 (2) ◽  
pp. 78
Author(s):  
Melissa Bello-Perez ◽  
Mikolaj Adamek ◽  
Julio Coll ◽  
Antonio Figueras ◽  
Beatriz Novoa ◽  
...  
Keyword(s):  
Gene Expression ◽  
Bacterial Infections ◽  
Quantitative Polymerase Chain Reaction ◽  
Tissue Expression ◽  
Interferon Response ◽  
Fish Skin ◽  
Tissue Expression Pattern ◽  
Chain Reaction ◽  
Polymerase Chain ◽  
Antiviral Role

Recent studies suggest that short pentraxins in fish might serve as biomarkers for not only bacterial infections, as in higher vertebrates including humans, but also for viral ones. These fish orthologs of mammalian short pentraxins are currently attracting interest because of their newly discovered antiviral activity. In the present work, the modulation of the gene expression of all zebrafish short pentraxins (CRP-like proteins, CRP1-7) was extensively analyzed by quantitative polymerase chain reaction. Initially, the tissue distribution of crp1-7 transcripts and how the transcripts varied in response to a bath infection with the spring viremia of carp virus, were determined. The expression of crp1-7 was widely distributed and generally increased after infection (mostly at 5 days post infection), except for crp1 (downregulated). Interestingly, several crp transcription levels significantly increased in skin. Further assays in mutant zebrafish of recombinant activation gene 1 (rag1) showed that all crps (except for crp2, downregulated) were already constitutively highly expressed in skin from rag1 knockouts and only increased moderately after viral infection. Similar results were obtained for most mx isoforms (a reporter gene of the interferon response), suggesting a general overcompensation of the innate immunity in the absence of the adaptive one.

Overlapping Allografts Provide Superior and More Reliable Surface Topography Matching Than Oblong Allografts: A Computer-Simulated Model Study

2021 ◽  
pp. 036354652110030
Author(s):  
Hailey P. Huddleston ◽  
Atsushi Urita ◽  
William M. Cregar ◽  
Theodore M. Wolfson ◽  
Brian J. Cole ◽  
...  
Keyword(s):  
Articular Cartilage ◽  
Surface Topography ◽  
Osteochondral Allograft ◽  
Cartilage Defects ◽  
Radius Of Curvature ◽  
Cartilage Surface ◽  
3 Dimensional ◽  
Cloud Models ◽  
And Cartilage ◽  
Osteochondral Allografts

Background: Osteochondral allograft transplantation is 1 treatment option for focal articular cartilage defects of the knee. Large irregular defects, which can be treated using an oblong allograft or multiple overlapping allografts, increase the procedure’s technical complexity and may provide suboptimal cartilage and subchondral surface matching between donor grafts and recipient sites. Purpose: To quantify and compare cartilage and subchondral surface topography mismatch and cartilage step-off for oblong and overlapping allografts using a 3-dimensional simulation model. Study Design: Controlled laboratory study. Methods: Human cadaveric medial femoral hemicondyles (n = 12) underwent computed tomography and were segmented into cartilage and bone components using 3-dimensional reconstruction and modeling software. Segments were then exported into point-cloud models. Modeled defect sizes of 17 × 30 mm were created on each recipient hemicondyle. There were 2 types of donor allografts from each condyle utilized: overlapping and oblong. Grafts were virtually harvested and implanted to optimally align with the defect to provide minimal cartilage surface topography mismatch. Least mean squares distances were used to measure cartilage and subchondral surface topography mismatch and cartilage step-off. Results: Cartilage and subchondral topography mismatch for the overlapping allograft group was 0.27 ± 0.02 mm and 0.80 ± 0.19 mm, respectively. In comparison, the oblong allograft group had significantly increased cartilage (0.62 ± 0.43 mm; P < .001) and subchondral (1.49 ± 1.10 mm; P < .001) mismatch. Cartilage step-off was also found to be significantly increased in the oblong group compared with the overlapping group ( P < .001). In addition, overlapping allografts more reliably provided a significantly higher percentage of clinically acceptable (0.5- and 1-mm thresholds) cartilage surface topography matching (overlapping: 100% for both 0.5 and 1 mm; oblong: 90% for 1 mm and 56% for 0.5 mm; P < .001) and cartilage step-off (overlapping: 100% for both 0.5 and 1 mm; oblong: 86% for 1 mm and 12% for 0.5 mm; P < .001). Conclusion: This computer simulation study demonstrated improved topography matching and decreased cartilage step-off with overlapping osteochondral allografts compared with oblong osteochondral allografts when using grafts from donors that were not matched to the recipient condyle by size or radius of curvature. These findings suggest that overlapping allografts may be superior in treating large, irregular osteochondral defects involving the femoral condyles with regard to technique. Clinical Relevance: This study suggests that overlapping allografts may provide superior articular cartilage surface topography matching compared with oblong allografts and do so in a more reliable fashion. Surgeons may consider overlapping allografts over oblong allografts because of the increased ease of topography matching during placement.

scholarly journals Use of Feldspathic Porcelain Veneers to Improve Smile Harmony: A 3-Year Follow-up Report

2016 ◽  
Vol 27 (6) ◽  
pp. 767-774 ◽  
Author(s):  
Leonardo Federizzi ◽  
Érica Alves Gomes ◽  
Samantha Schaffer Pugilato Báratro ◽  
Flares Baratto-Filho ◽  
Ataís Bacchi ◽  
...  
Keyword(s):  
Clinical Success ◽  
Relevant Literature ◽  
Clinical Results ◽  
Anterior Teeth ◽  
Correct Execution ◽  
Bond Interface ◽  
Laboratory Procedures ◽  
Feldspathic Porcelain ◽  
Strength And Durability

Abstract This case report describes an esthetic treatment to improve the shape and alignment of the anterior teeth, reestablishing smile harmony, using feldspathic porcelain veneers. Results of clinical follow up after 36 months are also presented. The advantages, disadvantages and limitations of the technique are detailed with reference to the relevant literature. This suggests that the success of treatment depends on adequate conditions of bonding between the veneers and the tooth complex, which involves parameters such as the strength and durability of the bond interface. Therefore, the clinical success of feldspathic porcelain veneers depends on the accurate selection of cases and correct execution of clinical and laboratory procedures. The rehabilitation involved from first right premolar to the left with feldspathic porcelain veneers made on refractory dies. After the 3-year follow up, excellent clinical results and patient satisfaction were achieved.

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