scholarly journals Hydrogel‐Guided, rAAV‐Mediated IGF‐I Overexpression Enables Long‐Term Cartilage Repair and Protection against Perifocal Osteoarthritis in a Large‐Animal Full‐Thickness Chondral Defect Model at One Year In Vivo

2021 ◽  
Vol 33 (16) ◽  
pp. 2008451
Author(s):  
Johanna Maihöfer ◽  
Henning Madry ◽  
Ana Rey‐Rico ◽  
Jagadeesh K. Venkatesan ◽  
Lars Goebel ◽  
...  
Keyword(s):  
Cartilage Repair ◽  
Large Animal ◽  
Full Thickness ◽  
Chondral Defect ◽  
Defect Model ◽  
Igf I ◽  
One Year

scholarly journals Functional regeneration and repair of tendons using biomimetic scaffolds loaded with recombinant periostin

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Yu Wang ◽  
Shanshan Jin ◽  
Dan Luo ◽  
Danqing He ◽  
Chunyan Shi ◽  
...  
Keyword(s):  
Progenitor Cell ◽  
Transcriptional Profiling ◽  
Collagen Scaffold ◽  
Defect Model ◽  
Tendon Regeneration ◽  
Biomimetic Scaffolds ◽  
Functional Regeneration

AbstractTendon injuries disrupt the balance between stability and mobility, causing compromised functions and disabilities. The regeneration of mature, functional tendons remains a clinical challenge. Here, we perform transcriptional profiling of tendon developmental processes to show that the extracellular matrix-associated protein periostin (Postn) contributes to the maintenance of tendon stem/progenitor cell (TSPC) functions and promotes tendon regeneration. We show that recombinant periostin (rPOSTN) promotes the proliferation and stemness of TSPCs, and maintains the tenogenic potentials of TSPCs in vitro. We also find that rPOSTN protects TSPCs against functional impairment during long-term passage in vitro. For in vivo tendon formation, we construct a biomimetic parallel-aligned collagen scaffold to facilitate TSPC tenogenesis. Using a rat full-cut Achilles tendon defect model, we demonstrate that scaffolds loaded with rPOSTN promote endogenous TSPC recruitment, tendon regeneration and repair with native-like hierarchically organized collagen fibers. Moreover, newly regenerated tendons show recovery of mechanical properties and locomotion functions.

scholarly journals Xenogenic Implantation of Equine Synovial Fluid-Derived Mesenchymal Stem Cells Leads to Articular Cartilage Regeneration

2018 ◽  
Vol 2018 ◽  
pp. 1-9 ◽  
Author(s):  
Mohammed Zayed ◽  
Steven Newby ◽  
Nabil Misk ◽  
Robert Donnell ◽  
Madhu Dhar
Keyword(s):  
Stem Cells ◽  
Mesenchymal Stem Cells ◽  
Stem Cell ◽  
Articular Cartilage ◽  
Synovial Fluid ◽  
Cartilage Repair ◽  
Cartilage Regeneration ◽  
Large Animal

Horses are widely used as large animal preclinical models for cartilage repair studies, and hence, there is an interest in using equine synovial fluid-derived mesenchymal stem cells (SFMSCs) in research and clinical applications. Since, we have previously reported that similar to bone marrow-derived MSCs (BMMSCs), SFMSCs may also exhibit donor-to-donor variations in their stem cell properties; the current study was carried out as a proof-of-concept study, to compare the in vivo potential of equine BMMSCs and SFMSCs in articular cartilage repair. MSCs from these two sources were isolated from the same equine donor. In vitro analyses confirmed a significant increase in COMP expression in SFMSCs at day 14. The cells were then encapsulated in neutral agarose scaffold constructs and were implanted into two mm diameter full-thickness articular cartilage defect in trochlear grooves of the rat femur. MSCs were fluorescently labeled, and one week after treatment, the knee joints were evaluated for the presence of MSCs to the injured site and at 12 weeks were evaluated macroscopically, histologically, and then by immunofluorescence for healing of the defect. The macroscopic and histological evaluations showed better healing of the articular cartilage in the MSCs’ treated knee than in the control. Interestingly, SFMSC-treated knees showed a significantly higher Col II expression, suggesting the presence of hyaline cartilage in the healed defect. Data suggests that equine SFMSCs may be a viable option for treating osteochondral defects; however, their stem cell properties require prior testing before application.

scholarly journals Autologous transplantation of canine long-term marrow culture cells genetically marked by retroviral vectors

Blood ◽  
1992 ◽  
Vol 79 (2) ◽  
pp. 356-364 ◽  
Author(s):  
RF Carter ◽  
AC Abrams-Ogg ◽  
JE Dick ◽  
SA Kruth ◽  
VE Valli ◽  
...  
Keyword(s):  
Gene Transfer ◽  
Autologous Transplantation ◽  
Large Animal Model ◽  
Multiple Infections ◽  
Large Animal ◽  
Retroviral Infection ◽  
Hematopoietic Stem ◽  
Marrow Cells

Abstract Retroviral infection of bone marrow cells in long-term marrow cultures (LTMCs) offers several theoretical advantages over other methods for gene transfer into hematopoietic stem cells. To investigate the feasibility of this approach in a large animal model system, we subjected LTMCs from nine dogs to multiple infections with retrovirus containing the neomycin phosphotransferase gene (neo) during 21 days of culture. Feeder layers, cocultivation, polycations, and selection were not used. The in vitro gene transfer efficiency was 70% as determined by polymerase chain reaction amplification of neo sequences in colony- forming unit granulocyte-macrophage (CFU-GM) obtained from day-21 LTMCs. Day-21 LTMC cells were infused into autologous recipients with (four dogs) and without (three dogs) marrow-ablative conditioning. At 3 months posttransplant, up to 10% of marrow cells contained the neo gene. This percentage declined to 0.1% to 1% at 10 to 21 months posttransplant. Neo was also detected in individual CFU-GM, burst- forming unit-erythroid (BFU-E), and CFU-Mix progenitors derived from marrow up to 21 months postinfusion and in cultures of peripheral blood- derived T cells up to 19 months postinfusion. There was no difference in the percentage of neo-marked cells present when dogs that received marrow ablative conditioning were compared with dogs receiving no conditioning. Detection of neo-marked marrow cells almost 2 years after autologous transplantation in a large mammalian species shows that retroviral infection of marrow cells in LTMCs is a potentially nontoxic and efficient protocol for gene transfer. Further, our results suggest that marrow conditioning and in vivo selection pressure to retain transplanted cells may not be absolute requirements for the retention of genetically marked cells in vivo.

scholarly journals Development and test for long-term stability of a synthetic standard for a quantitative Cryptosporidium parvum LightCycler™ PCR assay

2005 ◽  
Vol 3 (1) ◽  
pp. 15-25 ◽  
Author(s):  
Renata Filkorn-Kaiser ◽  
Konrad Botzenhart ◽  
Albrecht Wiedenmann
Keyword(s):  
Cryptosporidium Parvum ◽  
Surrogate Marker ◽  
Target Sequence ◽  
Positive Trend ◽  
Long Term Stability ◽  
Synthetic Standard ◽  
One Year

A recently described quantitative rapid cycle real time PCR (LightCycler™) assay detects Cryptosporidium parvum after in vitro excystation, which is a surrogate marker for the viability of the organisms. In the original assay the quantification standard is a dilution series of C. parvum oocysts with a microscopically determined excystation rate. The need to keep suspensions of viable oocysts in stock and to continuously monitor their excystation rate, however, renders the assay impracticable for routine application. A synthetic standard was developed to replace the in vivo standard and was calibrated using oocysts with known excystation rates. The standard consists of a 486 bp DNA segment ranging from 229 bp upstream to 79 bp downstream of the actual PCR target site. Aliquots of the standard were frozen and stored at −20 °C and at −70 °C or lyophilised and stored at room temperature in the dark. For a period of one year samples preserved with each of the three methods were restored every four or five weeks. They were amplified in the LightCycler™ and the crossing points (CP) were monitored. No significant trend in the raw CP values could be observed for any of the three storage methods. However, when the methods were compared to each other by calculating the CP ratios (−20 °C/−70 °C; −20 °C/lyophilised; −70 °C/lyophilised) at the 10 monitoring dates, the CP ratios −20 °C/−70 °C and −20 °C/lyophilised showed a highly significant positive trend (p<0.0001) while the CP ratio −70 °C/lyophilised did not differ from the null hypothesis (p=0.53). It can be concluded that the latter two preservation methods are both appropriate, while storage at −20 °C is less advisable. Calculations based on the molecular weight of the standard and on the assumption of an average yield of three sporozoites per oocyst led to the conclusion that the target sequence is probably located on a double copy gene

scholarly journals TCT-825 In-Vivo Long Term Evaluation of a Novel Mitral Valve Regurgitation Therapy: Experience in a Preclinical Large Animal Model

2013 ◽  
Vol 62 (18) ◽  
pp. B249
Author(s):  
Athanasios Peppas ◽  
Jon Wilson ◽  
Yanping Cheng ◽  
Christopher Seguin ◽  
Masahiko Shibuya ◽  
...  
Keyword(s):  
Animal Model ◽  
Mitral Valve ◽  
Mitral Valve Regurgitation ◽  
Large Animal Model ◽  
Large Animal ◽  
Valve Regurgitation ◽  
Term Evaluation

scholarly journals Multiphasic construct studied in an ectopic osteochondral defect model

2014 ◽  
Vol 11 (95) ◽  
pp. 20140184 ◽  
Author(s):  
June E. Jeon ◽  
Cédryck Vaquette ◽  
Christina Theodoropoulos ◽  
Travis J. Klein ◽  
Dietmar W. Hutmacher
Keyword(s):  
High Throughput ◽  
High Throughput Screening ◽  
Osteochondral Defect ◽  
Low Cost ◽  
Current Model ◽  
Large Animal ◽  
Micro Computed Tomography ◽  
Defect Model ◽  
Osteochondral Repair

In vivo osteochondral defect models predominantly consist of small animals, such as rabbits. Although they have an advantage of low cost and manageability, their joints are smaller and more easily healed compared with larger animals or humans. We hypothesized that osteochondral cores from large animals can be implanted subcutaneously in rats to create an ectopic osteochondral defect model for routine and high-throughput screening of multiphasic scaffold designs and/or tissue-engineered constructs (TECs). Bovine osteochondral plugs with 4 mm diameter osteochondral defect were fitted with novel multiphasic osteochondral grafts composed of chondrocyte-seeded alginate gels and osteoblast-seeded polycaprolactone scaffolds, prior to being implanted in rats subcutaneously with bone morphogenic protein-7. After 12 weeks of in vivo implantation, histological and micro-computed tomography analyses demonstrated that TECs are susceptible to mineralization. Additionally, there was limited bone formation in the scaffold. These results suggest that the current model requires optimization to facilitate robust bone regeneration and vascular infiltration into the defect site. Taken together, this study provides a proof-of-concept for a high-throughput osteochondral defect model. With further optimization, the presented hybrid in vivo model may address the growing need for a cost-effective way to screen osteochondral repair strategies before moving to large animal preclinical trials.

scholarly journals Activation of Peroxisome Proliferator-Activated Receptor γ (PPARγ) by Rosiglitazone Suppresses Components of the Insulin-Like Growth Factor Regulatory System in Vitro and in Vivo

Endocrinology ◽  
2007 ◽  
Vol 148 (2) ◽  
pp. 903-911 ◽  
Author(s):  
B. Lecka-Czernik ◽  
C. Ackert-Bicknell ◽  
M. L. Adamo ◽  
V. Marmolejos ◽  
G. A. Churchill ◽  
...  
Keyword(s):  
Regulatory System ◽  
Peroxisome Proliferator ◽  
Type I ◽  
Peroxisome Proliferator Activated Receptor ◽  
Igf I ◽  
Stromal Cell Line ◽  
Igf Receptor

Rosiglitazone (Rosi) belongs to the class of thiazolidinediones (TZDs) that are ligands for peroxisome proliferator-activated receptor γ (PPARγ). Stimulation of PPARγ suppresses bone formation and enhances marrow adipogenesis. We hypothesized that activation of PPARγ down-regulates components of the IGF regulatory system, leading to impaired osteoblast function. Rosi treatment (1 μm) of a marrow stromal cell line (UAMS-33) transfected with empty vector (U-33/c) or with PPARγ2 (U-33/γ2) were analyzed by microarray. Rosi reduced IGF-I, IGF-II, IGFBP-4, and the type I and II IGF receptor (IGF1R and IGF2R) expression at 72 h in U-33/γ2 compared with U-33/c cells (P < 0.01); these findings were confirmed by RT-PCR. Rosi reduced secreted IGF-I from U-33/γ2 cells by 75% (P < 0.05). Primary marrow stromal cells (MSCs) extracted from adult (8 months) and old (24 months) C57BL/6J (B6) mice were treated with Rosi (1 μm) for 48 h. IGF-I, IGFBP-4, and IGF1R transcripts were reduced in Rosi-treated MSCs compared with vehicle (P < 0.01) and secreted IGF-I was also suppressed (P < 0.05). B6 mice treated with Rosi (20 mg/kg·d) for short duration (i.e. 4 d), and long term (i.e. 7 wk) had reduced serum IGF-I; this was accompanied by markedly suppressed IGF-I transcripts in the liver and peripheral fat of treated animals. To determine whether Rosi affected circulating IGF-I in humans, we measured serum IGF-I, IGFBP-2, and IGFBP-3 at four time points in 50 postmenopausal women randomized to either Rosi (8 mg/d) or placebo. Rosi-treated subjects had significantly lower IGF-I at 8 wk than baseline (−25%, P < 0.05), and at 16 wk their levels were reduced 14% vs. placebo (P = 0.15). We conclude that Rosi suppresses IGF-I expression in bone and liver; these changes could affect skeletal acquisition through endocrine and paracrine pathways.

scholarly journals Autologous transplantation of canine long-term marrow culture cells genetically marked by retroviral vectors

Blood ◽  
1992 ◽  
Vol 79 (2) ◽  
pp. 356-364 ◽  
Author(s):  
RF Carter ◽  
AC Abrams-Ogg ◽  
JE Dick ◽  
SA Kruth ◽  
VE Valli ◽  
...  
Keyword(s):  
Gene Transfer ◽  
Autologous Transplantation ◽  
Large Animal Model ◽  
Multiple Infections ◽  
Large Animal ◽  
Retroviral Infection ◽  
Hematopoietic Stem ◽  
Marrow Cells

Retroviral infection of bone marrow cells in long-term marrow cultures (LTMCs) offers several theoretical advantages over other methods for gene transfer into hematopoietic stem cells. To investigate the feasibility of this approach in a large animal model system, we subjected LTMCs from nine dogs to multiple infections with retrovirus containing the neomycin phosphotransferase gene (neo) during 21 days of culture. Feeder layers, cocultivation, polycations, and selection were not used. The in vitro gene transfer efficiency was 70% as determined by polymerase chain reaction amplification of neo sequences in colony- forming unit granulocyte-macrophage (CFU-GM) obtained from day-21 LTMCs. Day-21 LTMC cells were infused into autologous recipients with (four dogs) and without (three dogs) marrow-ablative conditioning. At 3 months posttransplant, up to 10% of marrow cells contained the neo gene. This percentage declined to 0.1% to 1% at 10 to 21 months posttransplant. Neo was also detected in individual CFU-GM, burst- forming unit-erythroid (BFU-E), and CFU-Mix progenitors derived from marrow up to 21 months postinfusion and in cultures of peripheral blood- derived T cells up to 19 months postinfusion. There was no difference in the percentage of neo-marked cells present when dogs that received marrow ablative conditioning were compared with dogs receiving no conditioning. Detection of neo-marked marrow cells almost 2 years after autologous transplantation in a large mammalian species shows that retroviral infection of marrow cells in LTMCs is a potentially nontoxic and efficient protocol for gene transfer. Further, our results suggest that marrow conditioning and in vivo selection pressure to retain transplanted cells may not be absolute requirements for the retention of genetically marked cells in vivo.

Short-Term Hematopoietic Stem Cells (ST-HSC) Have Full Long-Term Capacity with Sustained but Reduced Potential Compared with LT-HSC.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 2550-2550
Author(s):  
Gerald A. Colvin ◽  
David Berz ◽  
Peter J. Quesenberry ◽  
Elaine Papa ◽  
Liansheng Liu
Keyword(s):  
Stem Cells ◽  
Stem Cell ◽  
Bone Marrow Cells ◽  
Conflicts Of Interest ◽  
Cell Number ◽  
Large Animal ◽  
Short Term ◽  
Hematopoietic Stem ◽  
One Year

Abstract Abstract 2550 Poster Board II-527 We evaluated the hypothesis that there was a homing defect between long-term (LT) hematopoietic stem cell (HSC) (KLS-Flk2-) and ST-HSC (KLS-Flk2+) that explained differences in engraftment potential and duration. Short-term HSC by definition have limited self-renewal capacity, generally described as giving rise to lymphohematopoiesis for 4–12 weeks before senescence. We performed three large animal engraftment studies into lethally ablated mice (950cGy split dose) looking at engraftment of both ST and LT-HSC cells delivered via intravenous, intraperitoneal and intra-femoral route. Two-hundred or 500 cells derived from B6/SJL mice were administered to each animal along with 300,000 recipient (C57/BLK) whole bone marrow cells for radioprotection following prior published studied [PNAS:98;14541, Stem Cells 24:1087] with optimization of flourochromes for better discrimination with our Cytopeia sorter. The animals were serially transplanted after eight months or one year to secondary recipients. In our hands, the ST-HSC engrafted animals did not lose chimerism over time. Review of the literature revealed that there were not confirmatory studies from those published from the initial one publication describing the ST-HSC. We found the ST-HSC were not short-term and persisted for one year in primary recipients and at least 3 months in secondary recipients. Engraftment kinetics favored LT-HSC over ST-HSC with engraftment examples at one year of 62% compared with 30% respectively when administered intravenously, 10% verses 4% given intra-femoral and 0.5% verse 0.3% given intraperitoneal. Chimerism was on average 50% better for the LT-HSC when compared with the ST-HSC and was irrespective of route proving that the differences seen are not due to homing deficiency but rather intrinsic differences in the two stem cell pools. Prior studies gave a maximum of 100 cells. Cell number was purposely increased for better differentiate of subtle differences in engraftment kinetics for statistical reasons. To avoid contamination of Flk2+ cells in the Flk2- cohort and vise-versa, discrimination of the gates were enhanced from that which was published prior. Double sorting of the cells confirmed that there was no appreciable cross contamination but obviously we cannot totally rule that out as a potentially confounding factor. In conclusion we found that ST-HSC as described have long-term capacity with intrinsic differences in engraftment potential that is not driven by a homing defect. Disclosures: No relevant conflicts of interest to declare.

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