Doxycycline preserves chondrocyte viability and function in human and calf articular cartilage ex vivo

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.

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Cadherins in early neural development

Cellular and Molecular Life Sciences ◽

10.1007/s00018-021-03815-9 ◽

2021 ◽

Author(s):

Karolina Punovuori ◽

Mattias Malaguti ◽

Sally Lowell

Keyword(s):

Adhesion Molecules ◽

Cell Fate ◽

Neural Development ◽

Ex Vivo ◽

Directed Differentiation ◽

Culture Dish ◽

Cell Identity ◽

Cell Fate Decisions ◽

Neural Tissues ◽

And Function

AbstractDuring early neural development, changes in signalling inform the expression of transcription factors that in turn instruct changes in cell identity. At the same time, switches in adhesion molecule expression result in cellular rearrangements that define the morphology of the emerging neural tube. It is becoming increasingly clear that these two processes influence each other; adhesion molecules do not simply operate downstream of or in parallel with changes in cell identity but rather actively feed into cell fate decisions. Why are differentiation and adhesion so tightly linked? It is now over 60 years since Conrad Waddington noted the remarkable "Constancy of the Wild Type” (Waddington in Nature 183: 1654–1655, 1959) yet we still do not fully understand the mechanisms that make development so reproducible. Conversely, we do not understand why directed differentiation of cells in a dish is sometimes unpredictable and difficult to control. It has long been suggested that cells make decisions as 'local cooperatives' rather than as individuals (Gurdon in Nature 336: 772–774, 1988; Lander in Cell 144: 955–969, 2011). Given that the cadherin family of adhesion molecules can simultaneously influence morphogenesis and signalling, it is tempting to speculate that they may help coordinate cell fate decisions between neighbouring cells in the embryo to ensure fidelity of patterning, and that the uncoupling of these processes in a culture dish might underlie some of the problems with controlling cell fate decisions ex-vivo. Here we review the expression and function of cadherins during early neural development and discuss how and why they might modulate signalling and differentiation as neural tissues are formed.

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271 Investigating the Relationship Between Nursery Pig Performance and Markers of Intestinal Morphology and Integrity

Journal of Animal Science ◽

10.1093/jas/skab054.161 ◽

2021 ◽

Vol 99 (Supplement_1) ◽

pp. 100-101

Author(s):

Carson M De Mille ◽

Nicholas K Gabler

Keyword(s):

Active Transport ◽

Feed Intake ◽

Ex Vivo ◽

Intestinal Morphology ◽

Ussing Chambers ◽

Nursery Pig ◽

Pig Performance ◽

Positive Correlations ◽

And Function ◽

Time Point

Abstract Weaning induces major structural and function changes to the small intestine of pigs and they transition from milk to solid feedstuffs. Thus, the objective of this study was to determine how intestinal morphology and function markers relate to feed intake and growth rates of nursery pig. Forty-eight weaned pigs (5.63 ± 0.50 kg) were randomly selected, individually penned and fed a common diet. Pig bodyweights and feed intake were determined at d 2, 7, and 21. At each time point, 16 pigs were randomly selected and euthanized. Sections of ileum were assessed for morphology [villus height (VH), crypt depth (CD) and VH:CD] and ex vivo transepithelial resistance (TER), macromolecule permeability (FD4), and active transport of glucose and glutamine via modified Ussing chambers. Within each period (d 0–2, 0–7, and 0–21), Pearson correlations were performed between ADG, ADFI, VH, VH:CD, TER, FD4 and active transport of glucose and glutamine. At d 2 post-weaning, no correlations (P > 0.05) were observed between performance and intestinal variables. By d 7, moderate positive correlations between VH and ADFI (r = 0.69, P = 0.005), VH and ADG (r = 0.68, P = 0.006) were reported. At 21 d post-weaning, moderate positive correlations were still observed for VH and ADFI (r = 0.55, P = 0.026) and between VH and ADG (r = 0.51, P = 0.042). Interestingly, ADFI and ADG tended to be negatively correlated with active glucose transport (r = -0.45, P = 0.083 and r = -0.47, P = 0.064, respectively) and active glutamine transport (r = -0.45, P = 0.083 and r = -0.46, P = 0.073, respectively). Markers of ileal integrity (TER and FD4) were not correlated with ADG or ADFI at any time point. Altogether, these data highlight the importance of intestinal morphology on early nursery pig performance.

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Optical Biomarkers for the Diagnosis of Osteoarthritis through Raman Spectroscopy: Radiological and Biochemical Validation Using Ex Vivo Human Cartilage Samples

Diagnostics ◽

10.3390/diagnostics11030546 ◽

2021 ◽

Vol 11 (3) ◽

pp. 546

Author(s):

Paula Casal-Beiroa ◽

Vanesa Balboa-Barreiro ◽

Natividad Oreiro ◽

Sonia Pértega-Díaz ◽

Francisco J. Blanco ◽

...

Keyword(s):

Raman Spectroscopy ◽

Articular Cartilage ◽

Ex Vivo ◽

Cartilage Degradation ◽

Calcium Pyrophosphate ◽

Calcium Pyrophosphate Dihydrate ◽

Label Free ◽

Molecular Fingerprint ◽

Human Cartilage ◽

Biochemical Validation

Osteoarthritis (OA) is the most common rheumatic disease, characterized by progressive articular cartilage degradation. Raman spectroscopy (RS) has been recently proposed as a label-free tool to detect molecular changes in musculoskeletal tissues. We used cartilage samples derived from human femoral heads to perform an ex vivo study of different Raman signals and ratios, related to major and minor molecular components of articular cartilage, hereby proposed as candidate optical biomarkers for OA. Validation was performed against the radiological Kellgren–Lawrence (K-L) grading system, as a gold standard, and cross-validated against sulfated glycosaminoglycans (sGAGs) and total collagens (Hyp) biochemical contents. Our results showed a significant decrease in sGAGs (SGAGs, A1063 cm−1/A1004 cm−1) and proteoglycans (PGs, A1375 cm−1/A1004 cm−1) and a significant increase in collagen disorganization (ColD/F, A1245 cm−1/A1270 cm−1), with OA severity. These were correlated with sGAGs or Hyp contents, respectively. Moreover, the SGAGs/HA ratio (A1063 cm−1/A960 cm−1), representing a functional matrix, rich in proteoglycans, to a mineralized matrix-hydroxyapatite (HA), was significantly lower in OA cartilage (K-L I vs. III–IV, p < 0.05), whilst the mineralized to collagenous matrix ratio (HA/Col, A960 cm−1/A920 cm−1) increased, being correlated with K-L. OA samples showed signs of tissue mineralization, supported by the presence of calcium crystals-related signals, such as phosphate, carbonate, and calcium pyrophosphate dihydrate (MGP, A960 cm−1/A1004 cm−1, MGC, A1070 cm−1/A1004 cm−1 and A1050 cm−1/A1004 cm−1). Finally, we observed an increase in lipids ratio (IL, A1450 cm−1/A1670 cm−1) with OA severity. As a conclusion, we have described the molecular fingerprint of hip cartilage, validating a panel of optical biomarkers and the potential of RS as a complementary diagnostic tool for OA.

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268 In-feed Antibiotics Elicit Intestinal Integrity Modifications Early in Post-weaning Life

Journal of Animal Science ◽

10.1093/jas/skab054.158 ◽

2021 ◽

Vol 99 (Supplement_1) ◽

pp. 99-99

Author(s):

Jessica M Johnson ◽

Emma T Helm ◽

Nicholas K Gabler ◽

Eric R Burrough ◽

Carson M De Mille

Keyword(s):

Growth Performance ◽

Fixed Effects ◽

Ex Vivo ◽

Phase 1 ◽

Physiological Mechanisms ◽

Nursery Pigs ◽

Ussing Chambers ◽

Intestinal Integrity ◽

And Function ◽

Dietary Treatments

Abstract The physiological mechanisms by which in-feed antibiotics improve pig growth performance are largely unknown. One proposed mode of action is improvements in intestinal integrity and function. Therefore, the objective of this study was to test the hypothesis that in-feed therapeutic and sub-therapeutic antibiotics would improve intestinal integrity and function in nursery pigs. Twenty-four weaned pigs (6.1±1.1 kg BW) were randomly allotted to individual pens and assigned one of three dietary treatments as follows (n = 8 pigs/trt): 1) control, no antibiotics (CON), 2) CON + sub-therapeutic chlortetracycline [40 ppm in feed (sCTC)], and 3) CON + chlortetracycline-tiamulin [400 ppm + 35 ppm, respectively (CTCDen)]. The study consisted of two consecutive 14 d phases. Chlortetracycline-tiamulin was only fed in phase 1, sCTC was fed in both phases. Phase 1 and 2 ADG, ADFI, and G:F were determined. After 28 d, ileal and colonic ex vivo intestinal integrity was assessed via transepithelial resistance (TER) and macromolecule flux (FD4) in modified Ussing chambers. All data were analyzed for the fixed effects of treatment and start BW as a covariate. In phase 1, compared with CON and sCTC, CTCDen tended to have greater ADG (0.28, 0.31, and 0.33 kg/d, respectively, P = 0.10) and ADFI (0.28, 0.30, and 0.35 kg/d, respectively, P = 0.09). No differences in phase 1 G:F were observed (P = 0.11). Phase 2 ADG, ADFI, and G:F did not differ (P > 0.10). Further, ileal TER and FD4 did not differ (P > 0.10). Colonic TER tended to be increased in sCTC compared with CON and CTCDen (78, 56, and 59 Ω/cm2, respectively, P = 0.07). Compared with CON, colonic FD4 flux was decreased in sCTC and CTCDen by 35–40% (P = 0.03). Altogether, these data indicate that in-feed antibiotics improve colon integrity early in production which may contribute to improved growth performance.

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Neurophysiological basis for neurogenic-mediated articular cartilage anabolism alteration

AJP Regulatory Integrative and Comparative Physiology ◽

10.1152/ajpregu.2001.280.1.r115 ◽

2001 ◽

Vol 280 (1) ◽

pp. R115-R122 ◽

Cited By ~ 7

Author(s):

Elvire Gouze-Decaris ◽

Lionel Philippe ◽

Alain Minn ◽

Philippe Haouzi ◽

Pierre Gillet ◽

...

Keyword(s):

Articular Cartilage ◽

Ex Vivo ◽

Cartilage Damage ◽

Intrathecal Injection ◽

Chronic Administration ◽

C Fibers ◽

Glutamatergic Synaptic Transmission ◽

C Fiber ◽

Fiber Stimulation ◽

Neurophysiological Basis

This study was designed to investigate the pathways involved in neurogenic-mediated articular cartilage damage triggered by a nonsystemic distant subcutaneous or intra-articular inflammation. The cartilage damage was assessed 24 h after subcutaneous or intra-articular complete Freund's adjuvant (CFA) injection measuring patellar proteoglycan (PG) synthesis (ex vivo [Na2 35SO4] incorporation) in 96 Wistar rats. Unilateral subcutaneous or intra-articular injection of CFA induced significant decrease (25–29%) in PG synthesis in both patellae. Chronic administration of capsaicin (50 mg · kg−1 · day−1 during 4 days), which blunted the normal response of C fiber stimulation, prevented the bilateral significant decrease in cartilage synthesis. Similarly, intrathecal injection of MK-801 (10 nmol/day during 5 days), which blocked the glutamatergic synaptic transmission at the dorsal horn of signal originating in primary afferent C fibers, eliminated the CFA-induced PG synthesis decrease in both patellae. Chemical sympathectomy, induced by guanethidine (12.5 mg · kg−1 · day−1 during 6 wk), also prevented PG synthesis alteration. Finally, compression of the spinal cord at the T3-T5 level had a similar protective effect on the reduction of [Na2 35SO4] incorporation. It is concluded that the signal that triggers articular cartilage synthesis damage induced by a distant local inflammation 1) is transmitted through the afferent C fibers, 2) makes glutamatergic synaptic connections with the preganglionic neurons of the sympathetic system, and 3) involves spinal and supraspinal pathways.

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Articular cartilage and the orthopaedic surgeon Part 1: Structure and function

Current Orthopaedics ◽

10.1016/0268-0890(94)90011-6 ◽

1994 ◽

Vol 8 (1) ◽

pp. 38-44 ◽

Cited By ~ 7

Author(s):

A.K. Jeffery

Keyword(s):

Articular Cartilage ◽

Structure And Function ◽

Orthopaedic Surgeon ◽

And Function

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hPSC-Derived Enteric Ganglioids Model Human ENS Development and Function

10.1101/2022.01.04.474746 ◽

2022 ◽

Author(s):

Homa Majd ◽

Ryan M Samuel ◽

Jonathan T Ramirez ◽

Ali Kalantari ◽

Kevin Barber ◽

...

Keyword(s):

Ex Vivo ◽

Xenograft Model ◽

Developmental Relationships ◽

Drug Candidates ◽

Effective Strategies ◽

Wide Range ◽

Functional Features ◽

And Function

The enteric nervous system (ENS) plays a central role in gut physiology and mediating the crosstalk between the gastrointestinal (GI) tract and other organs. The human ENS has remained elusive, highlighting the need for an in vitro modeling and mapping blueprint. Here we map out the developmental and functional features of the human ENS, by establishing robust and scalable 2D ENS cultures and 3D enteric ganglioids from human pluripotent stem cells (hPSCs). These models recapitulate the remarkable neuronal and glial diversity found in primary tissue and enable comprehensive molecular analyses that uncover functional and developmental relationships within these lineages. As a salient example of the power of this system, we performed in-depth characterization of enteric nitrergic neurons (NO neurons) which are implicated in a wide range of GI motility disorders. We conducted an unbiased screen and identified drug candidates that modulate the activity of NO neurons and demonstrated their potential in promoting motility in mouse colonic tissue ex vivo. We established a high-throughput strategy to define the developmental programs involved in NO neuron specification and discovered that PDGFR inhibition boosts the induction of NO neurons in enteric ganglioids. Transplantation of these ganglioids in the colon of NO neuron-deficient mice results in extensive tissue engraftment, providing a xenograft model for the study of human ENS in vivo and the development of cell-based therapies for neurodegenerative GI disorders. These studies provide a framework for deciphering fundamental features of the human ENS and designing effective strategies to treat enteric neuropathies.

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FLAMs: A self-replicating ex vivo model of alveolar macrophages for functional genetic studies

10.1101/2021.12.12.472259 ◽

2021 ◽

Author(s):

Sean Thomas ◽

Kathryn Wierenga ◽

James Pestka ◽

Andrew Olive

Keyword(s):

Alveolar Macrophages ◽

Ex Vivo ◽

Fetal Liver ◽

Expression Profiles ◽

Surface Expression ◽

Specific Gene ◽

Ex Vivo Model ◽

A Genome ◽

Underlying Mechanisms ◽

And Function

Alveolar macrophages (AMs) are tissue resident cells in the lungs derived from the fetal liver that maintain lung homeostasis and respond to inhaled stimuli. While the importance of AMs is undisputed, they remain refractory to standard experimental approaches and high-throughput functional genetics as they are challenging to isolate and rapidly lose AM properties in standard culture. This limitation hinders our understanding of key regulatory mechanisms that control AM maintenance and function. Here, we describe the development of a new model, fetal liver-derived alveolar-like macrophages (FLAMs), which maintains cellular morphologies, expression profiles, and functional mechanisms similar to murine AMs. FLAMs combine treatment with two key cytokines for AM maintenance, GM-CSF and TGFβ. We leveraged the long-term stability of FLAMs to develop functional genetic tools using CRISPR-Cas9-mediated gene editing. Targeted editing confirmed the role of AM-specific gene Marco and the IL-1 receptor Il1r1 in modulating the AM response to crystalline silica. Furthermore, a genome-wide knockout library using FLAMs identified novel genes required for surface expression of the AM marker Siglec-F, most notably those related to the peroxisome. Taken together, our results suggest that FLAMs are a stable, self-replicating model of AM function that enables previously impossible global genetic approaches to define the underlying mechanisms of AM maintenance and function.

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BHLHE40 Regulates IL-10 and IFN-γ Production in T Cells but Does Not Interfere With Human Type 1 Regulatory T Cell Differentiation

Frontiers in Immunology ◽

10.3389/fimmu.2021.683680 ◽

2021 ◽

Vol 12 ◽

Author(s):

Molly Javier Uyeda ◽

Robert A. Freeborn ◽

Brandon Cieniewicz ◽

Rosa Romano ◽

Ping (Pauline) Chen ◽

...

Keyword(s):

T Cells ◽

Cell Differentiation ◽

Ex Vivo ◽

Surface Expression ◽

Surface Molecules ◽

Ifn Γ ◽

And Function ◽

Tr1 Cells

Type 1 regulatory T (Tr1) cells are subset of peripherally induced antigen-specific regulatory T cells. IL-10 signaling has been shown to be indispensable for polarization and function of Tr1 cells. However, the transcriptional machinery underlying human Tr1 cell differentiation and function is not yet elucidated. To this end, we performed RNA sequencing on ex vivo human CD49b+LAG3+ Tr1 cells. We identified the transcription factor, BHLHE40, to be highly expressed in Tr1 cells. Even though Tr1 cells characteristically produce high levels of IL-10, we found that BHLHE40 represses IL-10 and increases IFN-γ secretion in naïve CD4+ T cells. Through CRISPR/Cas9-mediated knockout, we determined that IL10 significantly increased in the sgBHLHE40-edited cells and BHLHE40 is dispensable for naïve CD4+ T cells to differentiate into Tr1 cells in vitro. Interestingly, BHLHE40 overexpression induces the surface expression of CD49b and LAG3, co-expressed surface molecules attributed to Tr1 cells, but promotes IFN-γ production. Our findings uncover a novel mechanism whereby BHLHE40 acts as a regulator of IL-10 and IFN-γ in human CD4+ T cells.

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