38 research outputs found
Culture of equine fibroblast-like synoviocytes on synthetic tissue scaffolds towards meniscal tissue engineering: a preliminary cell-seeding study
Introduction. Tissue engineering is a new methodology for addressing meniscal injury or loss. Synovium may be an ideal source of cells for in vitro meniscal fibrocartilage formation, however, favorable in vitro culture conditions for synovium must be established in order to achieve this goal. The objective of this study was to determine cellularity, cell distribution, and extracellular matrix (ECM) formation of equine fibroblast-like synoviocytes (FLS) cultured on synthetic scaffolds, for potential application in synovium-based meniscal tissue engineering. Scaffolds included open-cell poly-L-lactic acid (OPLA) sponges and polyglycolic acid (PGA) scaffolds cultured in static and dynamic culture conditions, and PGA scaffolds coated in poly-L-lactic (PLLA) in dynamic culture conditions.Materials and Methods. Equine FLS were seeded on OPLA and PGA scaffolds, and cultured in a static environment or in a rotating bioreactor for 12 days. Equine FLS were also seeded on PGA scaffolds coated in 2% or 4% PLLA and cultured in a rotating bioreactor for 14 and 21 days. Three scaffolds from each group were fixed, sectioned and stained with Masson’s Trichrome, Safranin-O, and Hematoxylin and Eosin, and cell numbers and distribution were analyzed using computer image analysis. Three PGA and OPLA scaffolds from each culture condition were also analyzed for extracellular matrix (ECM) production via dimethylmethylene blue (sulfated glycosaminoglycan) assay and hydroxyproline (collagen) assay. PLLA coated PGA scaffolds were analyzed using double stranded DNA quantification as areflection of cellularity and confocal laser microscopy in a fluorescent cell viability assay.Results. The highest cellularity occurred in PGA constructs cultured in a rotating bioreactor, which also had a mean sulfated glycosaminoglycan content of 22.3 µg per scaffold. PGA constructs cultured in static conditions had the lowest cellularity. Cells had difficulty adhering to OPLA and the PLLA coating of PGA scaffolds; cellularity was inversely proportional to the concentration of PLLA used. PLLA coating did not prevent dissolution of the PGA scaffolds. All cell scaffold types and culture conditions produced non-uniform cellular distribution.Discussion/Conclusion. FLS-seeding of PGA scaffolds cultured in a rotating bioreactor resulted in the most optimal cell and matrix characteristics seen in this study. Cells grew only in the pores of the OPLA sponge, and could not adhere to the PLLA coating of PGA scaffold, due to the hydrophobic property of PLA. While PGA culture in a bioreactor produced measureable GAG, no culture technique produced visible collagen. For this reason, and due to the dissolution of PGA scaffolds, the culture conditions and scaffolds described here are not recommended for inducing fibrochondrogenesis in equine FLS for meniscal tissue engineering
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In vitro synthesis of tensioned synoviocyte bioscaffolds for meniscal fibrocartilage tissue engineering
BACKGROUND: Meniscal injury is a common cause of lameness in the dog. Tissue engineered bioscaffolds may be a treatment option for meniscal incompetency, and ideally would possess meniscus-like extracellular matrix (ECM) and withstand meniscal tensile hoop strains. Synovium may be a useful cell source for meniscal tissue engineering because of its natural role in meniscal deficiency and its in vitro chondrogenic potential. The objective of this study is to compare meniscal-like extracellular matrix content of hyperconfluent synoviocyte cell sheets (“HCS”) and hyperconfluent synoviocyte sheets which have been tensioned over wire hoops (tensioned synoviocyte bioscaffolds, “TSB”) and cultured for 1 month. RESULTS: Long term culture with tension resulted in higher GAG concentration, higher chondrogenic index, higher collagen concentration, and type II collagen immunoreactivity in TSB versus HCS. Both HCS and TSB were immunoreactive for type I collagen, however, HCS had mild, patchy intracellular immunoreactivity while TSB had diffuse moderate immunoreactivity over the entire bisocaffold. The tissue architecture was markedly different between TSB and HCS, with TSB containing collagen organized in bands and sheets. Both HCS and TSB expressed alpha smooth muscle actin and displayed active contractile behavior. Double stranded DNA content was not different between TSB and HCS, while cell viability decreased in TSB. CONCLUSIONS: Long term culture of synoviocytes with tension improved meniscal-like extra cellular matrix components, specifically, the total collagen content, including type I and II collagen, and increased GAG content relative to HCS. Future research is warranted to investigate the potential of TSB for meniscal tissue engineering
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The effect of agility exercise on eicosanoid excretion, oxidant status, and plasma lactate in dogs
urinary eicosanoids, urinary 15F[subscript2t]-isoprostane (lipid peroxidation marker) and hematological/biochemical changes in plasma. Fifteen adult dogs had blood and urine samples obtained prior to, immediately and 4-hours following an agility exercise.
Results: Hematocrit, red blood cells (RBC), albumin, and hemoglobin increased following exercise, with greatest increases correlating to increased skill group (novice, intermediate, masters); at 4-hours post-exercise, hematocrit, RBC, and hemoglobin were decreased. Phosphorus increased following exercise with the greatest increase in novice and intermediates. Plasma lactate increased 3.6-fold in masters, 3.2-fold in intermediates, and 1.2-fold in novice dogs. Urine thromboxane B-2 (TXB2) more than tripled 4-hours post-exercise while 6-keto prostaglandin F[subscript 1 alpha] (PGF1 alpha, prostacyclin metabolite), prostaglandin E[subscript 2] metabolites (13,14-dihydro-15-keto-prostaglandin A[subscript 2] and 13, 14-dihydro-15-keto-prostaglandin E[subscript 2], and 13,14-dihydro-15-keto prostaglandin F[subscript 2 alpha] were unaffected as determined by a competitive enzyme immunoassay and standardized by division with urine creatinine. Urine 15F[subscript 2t]-isoprostane increased insignificantly.
Conclusions: Alterations in the plasma post-exercise were likely due to hemoconcentration from insensible water loss, splenic contraction and sympathetic stimulation while 4-hours later autohemodilution reduced RBC parameters. Elevations in plasma lactate and urinary TXB2 correlated with advanced skill level/speed of the dogs.Keywords: Oxidant stress, Agility exercise, Urinary eicosanoids, Thromboxane B[subscript 2], Dog, Plasma lactate, Urinary isoprostan
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Culture of equine fibroblast-like synoviocytes on synthetic tissue scaffolds towards meniscal tissue engineering: a preliminary cell-seeding study
INTRODUCTION: Tissue engineering is a new methodology for addressing meniscal
injury or loss. Synovium may be an ideal source of cells for in vitro meniscal fibrocartilage
formation, however, favorable in vitro culture conditions for synovium must be
established in order to achieve this goal. The objective of this study was to determine
cellularity, cell distribution, and extracellular matrix (ECM) formation of equine
fibroblast-like synoviocytes (FLS) cultured on synthetic scaffolds, for potential application
in synovium-based meniscal tissue engineering. Scaffolds included open-cell
poly-L-lactic acid (OPLA) sponges and polyglycolic acid (PGA) scaffolds cultured
in static and dynamic culture conditions, and PGA scaffolds coated in poly-L-lactic
(PLLA) in dynamic culture conditions.
MATERIALS AND METHODS: Equine FLS were seeded on OPLA and PGA scaffolds, and
cultured in a static environment or in a rotating bioreactor for 12 days. Equine FLS
were also seeded on PGA scaffolds coated in 2% or 4% PLLA and cultured in a
rotating bioreactor for 14 and 21 days. Three scaffolds from each group were fixed,
sectioned and stained with Masson’s Trichrome, Safranin-O, and Hematoxylin and
Eosin, and cell numbers and distribution were analyzed using computer image analysis.
Three PGA and OPLA scaffolds from each culture condition were also analyzed
for extracellular matrix (ECM) production via dimethylmethylene blue (sulfated
glycosaminoglycan) assay and hydroxyproline (collagen) assay. PLLA coated PGA
scaffolds were analyzed using double stranded DNA quantification as a reflection of
cellularity and confocal laser microscopy in a fluorescent cell viability assay.
RESULTS: The highest cellularity occurred in PGA constructs cultured in a rotating
bioreactor, which also had a mean sulfated glycosaminoglycan content of 22.3 μg per
scaffold. PGA constructs cultured in static conditions had the lowest cellularity. Cells
had difficulty adhering to OPLA and the PLLA coating of PGA scaffolds; cellularity
was inversely proportional to the concentration of PLLA used. PLLA coating did not
prevent dissolution of the PGA scaffolds. All cell scaffold types and culture conditions
produced non-uniformcellular distribution. DISCUSSION/CONCLUSION: FLS-seeding of PGA scaffolds cultured in a rotating bioreactor resulted in the most optimal cell and matrix characteristics seen in this study.
Cells grew only in the pores of the OPLA sponge, and could not adhere to the PLLA
coating of PGA scaffold, due to the hydrophobic property of PLA. While PGA culture
in a bioreactor produced measureable GAG, no culture technique produced
visible collagen. For this reason, and due to the dissolution of PGA scaffolds, the
culture conditions and scaffolds described here are not recommended for inducing
fibrochondrogenesis in equine FLS for meniscal tissue engineering.Keywords: Stifle,
Orthopedics,
Bioreactors,
Fibroblast-like synoviocytes,
Meniscus,
Cell scaffolds,
Tissue engineering,
Bioengineering,
Veterinary Medicine,
Surgery and Surgical Specialties,
EquineThis is the publisher’s final pdf. The published article is copyrighted by the author(s) and published by PeerJ. The published article can be found at: https://peerj.com/
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Effect of Hydrocortisone on Mortality and Organ Support in Patients With Severe COVID-19: The REMAP-CAP COVID-19 Corticosteroid Domain Randomized Clinical Trial.
Importance: Evidence regarding corticosteroid use for severe coronavirus disease 2019 (COVID-19) is limited. Objective: To determine whether hydrocortisone improves outcome for patients with severe COVID-19. Design, Setting, and Participants: An ongoing adaptive platform trial testing multiple interventions within multiple therapeutic domains, for example, antiviral agents, corticosteroids, or immunoglobulin. Between March 9 and June 17, 2020, 614 adult patients with suspected or confirmed COVID-19 were enrolled and randomized within at least 1 domain following admission to an intensive care unit (ICU) for respiratory or cardiovascular organ support at 121 sites in 8 countries. Of these, 403 were randomized to open-label interventions within the corticosteroid domain. The domain was halted after results from another trial were released. Follow-up ended August 12, 2020. Interventions: The corticosteroid domain randomized participants to a fixed 7-day course of intravenous hydrocortisone (50 mg or 100 mg every 6 hours) (n = 143), a shock-dependent course (50 mg every 6 hours when shock was clinically evident) (n = 152), or no hydrocortisone (n = 108). Main Outcomes and Measures: The primary end point was organ support-free days (days alive and free of ICU-based respiratory or cardiovascular support) within 21 days, where patients who died were assigned -1 day. The primary analysis was a bayesian cumulative logistic model that included all patients enrolled with severe COVID-19, adjusting for age, sex, site, region, time, assignment to interventions within other domains, and domain and intervention eligibility. Superiority was defined as the posterior probability of an odds ratio greater than 1 (threshold for trial conclusion of superiority >99%). Results: After excluding 19 participants who withdrew consent, there were 384 patients (mean age, 60 years; 29% female) randomized to the fixed-dose (n = 137), shock-dependent (n = 146), and no (n = 101) hydrocortisone groups; 379 (99%) completed the study and were included in the analysis. The mean age for the 3 groups ranged between 59.5 and 60.4 years; most patients were male (range, 70.6%-71.5%); mean body mass index ranged between 29.7 and 30.9; and patients receiving mechanical ventilation ranged between 50.0% and 63.5%. For the fixed-dose, shock-dependent, and no hydrocortisone groups, respectively, the median organ support-free days were 0 (IQR, -1 to 15), 0 (IQR, -1 to 13), and 0 (-1 to 11) days (composed of 30%, 26%, and 33% mortality rates and 11.5, 9.5, and 6 median organ support-free days among survivors). The median adjusted odds ratio and bayesian probability of superiority were 1.43 (95% credible interval, 0.91-2.27) and 93% for fixed-dose hydrocortisone, respectively, and were 1.22 (95% credible interval, 0.76-1.94) and 80% for shock-dependent hydrocortisone compared with no hydrocortisone. Serious adverse events were reported in 4 (3%), 5 (3%), and 1 (1%) patients in the fixed-dose, shock-dependent, and no hydrocortisone groups, respectively. Conclusions and Relevance: Among patients with severe COVID-19, treatment with a 7-day fixed-dose course of hydrocortisone or shock-dependent dosing of hydrocortisone, compared with no hydrocortisone, resulted in 93% and 80% probabilities of superiority with regard to the odds of improvement in organ support-free days within 21 days. However, the trial was stopped early and no treatment strategy met prespecified criteria for statistical superiority, precluding definitive conclusions. Trial Registration: ClinicalTrials.gov Identifier: NCT02735707
Effect of angiotensin-converting enzyme inhibitor and angiotensin receptor blocker initiation on organ support-free days in patients hospitalized with COVID-19
IMPORTANCE Overactivation of the renin-angiotensin system (RAS) may contribute to poor clinical outcomes in patients with COVID-19.
Objective To determine whether angiotensin-converting enzyme (ACE) inhibitor or angiotensin receptor blocker (ARB) initiation improves outcomes in patients hospitalized for COVID-19.
DESIGN, SETTING, AND PARTICIPANTS In an ongoing, adaptive platform randomized clinical trial, 721 critically ill and 58 non–critically ill hospitalized adults were randomized to receive an RAS inhibitor or control between March 16, 2021, and February 25, 2022, at 69 sites in 7 countries (final follow-up on June 1, 2022).
INTERVENTIONS Patients were randomized to receive open-label initiation of an ACE inhibitor (n = 257), ARB (n = 248), ARB in combination with DMX-200 (a chemokine receptor-2 inhibitor; n = 10), or no RAS inhibitor (control; n = 264) for up to 10 days.
MAIN OUTCOMES AND MEASURES The primary outcome was organ support–free days, a composite of hospital survival and days alive without cardiovascular or respiratory organ support through 21 days. The primary analysis was a bayesian cumulative logistic model. Odds ratios (ORs) greater than 1 represent improved outcomes.
RESULTS On February 25, 2022, enrollment was discontinued due to safety concerns. Among 679 critically ill patients with available primary outcome data, the median age was 56 years and 239 participants (35.2%) were women. Median (IQR) organ support–free days among critically ill patients was 10 (–1 to 16) in the ACE inhibitor group (n = 231), 8 (–1 to 17) in the ARB group (n = 217), and 12 (0 to 17) in the control group (n = 231) (median adjusted odds ratios of 0.77 [95% bayesian credible interval, 0.58-1.06] for improvement for ACE inhibitor and 0.76 [95% credible interval, 0.56-1.05] for ARB compared with control). The posterior probabilities that ACE inhibitors and ARBs worsened organ support–free days compared with control were 94.9% and 95.4%, respectively. Hospital survival occurred in 166 of 231 critically ill participants (71.9%) in the ACE inhibitor group, 152 of 217 (70.0%) in the ARB group, and 182 of 231 (78.8%) in the control group (posterior probabilities that ACE inhibitor and ARB worsened hospital survival compared with control were 95.3% and 98.1%, respectively).
CONCLUSIONS AND RELEVANCE In this trial, among critically ill adults with COVID-19, initiation of an ACE inhibitor or ARB did not improve, and likely worsened, clinical outcomes.
TRIAL REGISTRATION ClinicalTrials.gov Identifier: NCT0273570
Fibrochondrogenic potential of synoviocytes from osteoarthritic and normal joints cultured as tensioned bioscaffolds for meniscal tissue engineering in dogs
Meniscal tears are a common cause of stifle lameness in dogs. Use of autologous synoviocytes from the affected stifle is an attractive cell source for tissue engineering replacement fibrocartilage. However, the diseased state of these cells may impede in vitro fibrocartilage formation. Synoviocytes from 12 osteoarthritic (“oaTSB”) and 6 normal joints (“nTSB”) were cultured as tensioned bioscaffolds and compared for their ability to synthesize fibrocartilage sheets. Gene expression of collagens type I and II were higher and expression of interleukin-6 was lower in oaTSB versus nTSB. Compared with nTSB, oaTSB had more glycosaminoglycan and alpha smooth muscle staining and less collagen I and II staining on histologic analysis, whereas collagen and glycosaminoglycan quantities were similar. In conclusion, osteoarthritic joint—origin synoviocytes can produce extracellular matrix components of meniscal fibrocartilage at similar levels to normal joint—origin synoviocytes, which makes them a potential cell source for canine meniscal tissue engineering
In vitro synthesis of tensioned synoviocyte bioscaffolds for meniscal fibrocartilage tissue engineering
BACKGROUND: Meniscal injury is a common cause of lameness in the dog. Tissue engineered bioscaffolds may be a treatment option for meniscal incompetency, and ideally would possess meniscus- like extracellular matrix (ECM) and withstand meniscal tensile hoop strains. Synovium may be a useful cell source for meniscal tissue engineering because of its natural role in meniscal deficiency and its in vitro chondrogenic potential. The objective of this study is to compare meniscal -like extracellular matrix content of hyperconfluent synoviocyte cell sheets (“HCS”) and hyperconfluent synoviocyte sheets which have been tensioned over wire hoops (tensioned synoviocyte bioscaffolds, “TSB”) and cultured for 1 month. RESULTS: Long term culture with tension resulted in higher GAG concentration, higher chondrogenic index, higher collagen concentration, and type II collagen immunoreactivity in TSB versus HCS. Both HCS and TSB were immunoreactive for type I collagen, however, HCS had mild, patchy intracellular immunoreactivity while TSB had diffuse moderate immunoreactivity over the entire bisocaffold. The tissue architecture was markedly different between TSB and HCS, with TSB containing collagen organized in bands and sheets. Both HCS and TSB expressed alpha smooth muscle actin and displayed active contractile behavior. Double stranded DNA content was not different between TSB and HCS, while cell viability decreased in TSB. CONCLUSIONS: Long term culture of synoviocytes with tension improved meniscal- like extra cellular matrix components, specifically, the total collagen content, including type I and II collagen, and increased GAG content relative to HCS. Future research is warranted to investigate the potential of TSB for meniscal tissue engineering
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Fibrochondrogenic potential of synoviocytes from osteoarthritic and normal joints cultured as tensioned bioscaffolds for meniscal tissue engineering in dogs
Meniscal tears are a common cause of stifle lameness in dogs. Use of autologous
synoviocytes from the affected stifle is an attractive cell source for tissue engineering
replacement fibrocartilage. However, the diseased state of these cells may impede
in vitro fibrocartilage formation. Synoviocytes from 12 osteoarthritic (“oaTSB”) and
6 normal joints (“nTSB”) were cultured as tensioned bioscaffolds and compared for
their ability to synthesize fibrocartilage sheets. Gene expression of collagens type
I and II were higher and expression of interleukin-6 was lower in oaTSB versus
nTSB. Compared with nTSB, oaTSB had more glycosaminoglycan and alpha smooth
muscle staining and less collagen I and II staining on histologic analysis, whereas
collagen and glycosaminoglycan quantities were similar. In conclusion, osteoarthritic
joint—origin synoviocytes can produce extracellular matrix components of meniscal
fibrocartilage at similar levels to normal joint—origin synoviocytes, which makes
them a potential cell source for canine meniscal tissue engineering.This is the publisher’s final pdf. The published article is copyrighted by the author(s) and published by PeerJ. The published article can be found at: https://peerj.com/.Keywords: Tissue engineering, Bioengineering, Cell culture, Meniscus, Veterinary Medicine, Osteoarthritis, Cell Biology, Synovium, Orthopedic