Maintenance of connective tissue integrity is fundamental to sustain function, requiring protein turnover to repair damaged tissue. However, connective tissue proteome dynamics remain largely ...undefined, as do differences in turnover rates of individual proteins in the collagen and glycoprotein phases of connective tissue extracellular matrix (ECM). Here, we investigate proteome dynamics in the collagen and glycoprotein phases of connective tissues by exploiting the spatially distinct fascicular (collagen-rich) and interfascicular (glycoprotein-rich) ECM phases of tendon. Using isotope labelling, mass spectrometry and bioinformatics, we calculate turnover rates of individual proteins within rat Achilles tendon and its ECM phases. Our results demonstrate complex proteome dynamics in tendon, with ~1000 fold differences in protein turnover rates, and overall faster protein turnover within the glycoprotein-rich interfascicular matrix compared to the collagen-rich fascicular matrix. These data provide insights into the complexity of proteome dynamics in tendon, likely required to maintain tissue homeostasis.
Tendon injuries are a common age-related degenerative condition where current treatment strategies fail to restore functionality and normal quality of life. This disease also occurs naturally in ...horses, with many similarities to human tendinopathy making it an ideal large animal model for human disease. Regenerative approaches are increasingly used to improve outcome involving mesenchymal stem cells (MSCs), supported by clinical data where injection of autologous bone marrow derived MSCs (BM-MSCs) suspended in marrow supernatant into injured tendons has halved the re-injury rate in racehorses. We hypothesized that stem cell therapy induces a matrix more closely resembling normal tendon than the fibrous scar tissue formed by natural repair. Twelve horses with career-ending naturally-occurring superficial digital flexor tendon injury were allocated randomly to treatment and control groups. 1X10(7) autologous BM-MSCs suspended in 2 ml of marrow supernatant were implanted into the damaged tendon of the treated group. The control group received the same volume of saline. Following a 6 month exercise programme horses were euthanized and tendons assessed for structural stiffness by non-destructive mechanical testing and for morphological and molecular composition. BM-MSC treated tendons exhibited statistically significant improvements in key parameters compared to saline-injected control tendons towards that of normal tendons and those in the contralateral limbs. Specifically, treated tendons had lower structural stiffness (p<0.05) although no significant difference in calculated modulus of elasticity, lower (improved) histological scoring of organisation (p<0.003) and crimp pattern (p<0.05), lower cellularity (p<0.007), DNA content (p<0.05), vascularity (p<0.03), water content (p<0.05), GAG content (p<0.05), and MMP-13 activity (p<0.02). Treatment with autologous MSCs in marrow supernatant therefore provides significant benefits compared to untreated tendon repair in enhancing normalisation of biomechanical, morphological, and compositional parameters. These data in natural disease, with no adverse findings, support the use of this treatment for human tendon injuries.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Rapid developments in Regenerative Medicine and Tissue Engineering has witnessed an increasing drive toward clinical translation of breakthrough technologies. However, the progression of promising ...preclinical data to achieve successful clinical market authorisation remains a bottleneck. One hurdle for progress to the clinic is the transition from small animal research to advanced preclinical studies in large animals to test safety and efficacy of products. Notwithstanding this, to draw meaningful and reliable conclusions from animal experiments it is critical that the species and disease model of choice is relevant to answer the research question as well as the clinical problem. Selecting the most appropriate animal model requires in-depth knowledge of specific species and breeds to ascertain the adequacy of the model and outcome measures that closely mirror the clinical situation. Traditional reductionist approaches in animal experiments, which often do not sufficiently reflect the studied disease, are still the norm and can result in a disconnect in outcomes observed between animal studies and clinical trials. To address these concerns a reconsideration in approach will be required. This should include a stepwise approach using
experiments as well as
modeling to minimize the need for
studies for screening and early development studies, followed by large animal models which more closely resemble human disease. Naturally occurring, or spontaneous diseases in large animals remain a largely untapped resource, and given the similarities in pathophysiology to humans they not only allow for studying new treatment strategies but also disease etiology and prevention. Naturally occurring disease models, particularly for longer lived large animal species, allow for studying disorders at an age when the disease is most prevalent. As these diseases are usually also a concern in the chosen veterinary species they would be beneficiaries of newly developed therapies. Improved awareness of the progress in animal models is mutually beneficial for animals, researchers, human and veterinary patients. In this overview we describe advantages and disadvantages of various animal models including domesticated and companion animals used in regenerative medicine and tissue engineering to provide an informed choice of disease-relevant animal models.
Articular cartilage displays a poor repair capacity. The aim of cell-based therapies for cartilage defects is to repair damaged joint surfaces with a functional replacement tissue. Currently, ...chondrocytes removed from a healthy region of the cartilage are used but they are unable to retain their phenotype in expanded culture. The resulting repair tissue is fibrocartilaginous rather than hyaline, potentially compromising long-term repair. Mesenchymal stem cells, particularly bone marrow stromal cells (BMSC), are of interest for cartilage repair due to their inherent replicative potential. However, chondrocyte differentiated BMSCs display an endochondral phenotype, that is, can terminally differentiate and form a calcified matrix, leading to failure in long-term defect repair. Here, we investigate the isolation and characterisation of a human cartilage progenitor population that is resident within permanent adult articular cartilage.
Human articular cartilage samples were digested and clonal populations isolated using a differential adhesion assay to fibronectin. Clonal cell lines were expanded in growth media to high population doublings and karyotype analysis performed. We present data to show that this cell population demonstrates a restricted differential potential during chondrogenic induction in a 3D pellet culture system. Furthermore, evidence of high telomerase activity and maintenance of telomere length, characteristic of a mesenchymal stem cell population, were observed in this clonal cell population. Lastly, as proof of principle, we carried out a pilot repair study in a goat in vivo model demonstrating the ability of goat cartilage progenitors to form a cartilage-like repair tissue in a chondral defect.
In conclusion, we propose that we have identified and characterised a novel cartilage progenitor population resident in human articular cartilage which will greatly benefit future cell-based cartilage repair therapies due to its ability to maintain chondrogenicity upon extensive expansion unlike full-depth chondrocytes that lose this ability at only seven population doublings.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Understanding the high temperature behavior of collagen and collagenous tissue is important for surgical procedures and biomaterials processing for the food, pharmaceutical, and cosmetics industries. ...One primary event for proteins is thermal denaturation that involves unfolding the polypeptide chains while maintaining the primary structure intact. Collagen in the extracellular matrix of cartilage and other connective tissue is a hierarchical material containing bundles of triple‐helical fibers associated with water and proteoglycan components. Thermal analysis of dehydrated collagen indicates irreversible denaturation at high temperature between 135°C and 200°C, with another reversible event at ∼60‐80°C for hydrated samples. We report high temperature Raman spectra for freeze‐dried cartilage samples that show an increase in laser‐excited fluorescence interpreted as conformational changes associated with denaturation above 140°C. Spectra for separated collagen and proteoglycan fractions extracted from cartilage indicate the changes are associated with collagen. The Raman data also show appearance of new features indicating peptide bond hydrolysis at high temperature implying that molecular H2O is retained within the freeze‐dried tissue. This is confirmed by thermogravimetric analysis that show 5‐7 wt% H2O remaining within freeze‐dried cartilage that is released progressively upon heating up to 200°C. Spectra obtained after exposure to high temperature and re‐hydration following recovery indicate that the capacity of the denatured collagen to re‐absorb water is reduced. Our results are important for revealing the presence of bound H2O within the collagen component of connective tissue even after freeze‐drying and its role in denaturation that is accompanied by or perhaps preceded by breakdown of the primary polypeptide structure.
The diagnosis of tendon injury relies on clinical signs and diagnostic imaging but imaging is subjective and does not always correlate with clinical signs. A molecular marker would potentially offer ...a sensitive and specific diagnostic tool that could also provide objective assessment of healing for the comparison of different treatments. Cartilage Oligomeric Matrix Protein (COMP) has been used as a molecular marker for osteoarthritis in humans and horses but assays for the protein in tendon sheath synovial fluids have shown overlap between horses affected by tendinopathy and controls. We hypothesized that quantifying a COMP neoepitope would be more discriminatory of injury. COMP fragments were purified from synovial fluids of horses with intra-thecal tendon injuries and media from equine tendon explants, and mass spectrometry of a consistent and abundant fragment revealed a ~100 kDa COMP fragment with a new N-terminus at the 78th amino-acid (NH
-TPRVSVRP) located just outside the junctional region of the protein. A competitive inhibition ELISA based on a polyclonal antibody raised to this sequence yielded more than a 10-fold rise in the mean neoepitope levels for tendinopathy cases compared to controls (5.3 ± 1.3 µg/mL (
= 7) versus 58.8 ± 64.3 µg/mL (
= 13);
= 0.002). However, there was some cross-reactivity of the neoepitope polyclonal antiserum with intact COMP, which could be blocked by a peptide spanning the neoepitope. The modified assay demonstrated a lower concentration but a significant > 500-fold average rise with tendon injury (2.5 ± 2.2 ng/mL (
= 6) versus 1029.8 ± 2188.8 ng/ml (
= 14);
= 0.013). This neo-epitope assay therefore offers a potentially useful marker for clinical use.
Rotator cuff tendon repair in humans is a commonly performed procedure aimed at restoring the tendon-bone interface. Despite significant innovation of surgical techniques and suture anchor implants, ...only 60% of repairs heal successfully. One strategy to enhance repair is the use of bioactive sutures that provide the native tendon with biophysical cues for healing. We investigated the tissue response to a multifilament electrospun polydioxanone (PDO) suture in a sheep tendon injury model characterised by a natural history of failure of healing.
Eight skeletally mature English Mule sheep underwent repair with electrospun sutures. Monofilament sutures were used as a control. Three months after surgery, all tendon repairs healed, without systemic features of inflammation, signs of tumour or infection at necropsy. A mild local inflammatory reaction was seen. On histology the electrospun sutures were densely infiltrated with predominantly tendon fibroblast-like cells. In comparison, no cellular infiltration was observed in the control suture. Neovascularisation was observed within the electrospun suture, whilst none was seen in the control. Foreign body giant cells were rarely seen with either sutures.
This study demonstrates that a tissue response can be induced in tendon with a multifilament electrospun suture with no safety concerns.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
We investigated endogenous tissue response to a woven and electrospun polydioxanone (PDO) and polycaprolactone (PCL) patch intended for tendon repair. A sheep tendon injury model characterised by a ...natural history of consistent failure of healing was chosen to assess the biological potential of woven and aligned electrospun fibres to induce a reparative response. Patches were implanted into 8 female adult English Mule sheep. Significant infiltration of tendon fibroblasts was observed within the electrospun component of the patch but not within the woven component. The cellular infiltrate into the electrospun fibres was accompanied by an extensive network of new blood vessel formation. Tendon fibroblasts were the most abundant scaffold-populating cell type. CD45
, CD4
and CD14
cells were also present, with few foreign body giant cells. There were no local or systemic signs of excessive inflammation with normal hematology and serology for inflammatory markers three months after scaffold implantation. In conclusion, we demonstrate that an endogenous healing response can be safely induced in tendon by means of biophysical cues using a woven and electrospun patch.
Resveratrol (RSV; trans-3,5,4′-trihydroxystilbene) strongly activates sirtuin 1, and it and its analogue V29 enhance the proliferation of mesenchymal stem/stromal cells (MSCs).Although culture medium ...containing 5‐azacytydine and RSV inhibits senescence of adipose tissue–derived MSCs isolated from horses with metabolic syndrome, few studies have reported the effects of RSV on equine bone marrow–derived MSCs (eBMMSCs) isolated from horses without metabolic syndrome. The aim of this study was to investigate the effects of RSV and V29 on the cell cycle of eBMMSCs. Following treatment with 5 µM RSV or 10 µM V29, the cell proliferation capacity of eBMMSCs derived from seven horses was evaluated by EdU (5-ethynyl-2′-deoxyuridine) and Ki-67 antibody assays. Brightfield images of cells and immunofluorescent images of EdU, Ki-67, and DAPI staining were recorded by fluorescence microscopy, and the number of cells positive for each was quantified and compared by Friedman’s test at P<0.05. The growth fraction of eBMMSCs was significantly increased by RSV and V29 as measured by the EdU assay (control 28.1% ± 13.8%, V29 31.8% ± 14.6%, RSV 32.0% ± 10.8%; mean ± SD; P<0.05) but not as measured by the Ki-67 antibody assay (control 27.0% ± 11.2%, V29 27.4% ± 10.8%, RSV 27.7% ± 6.8%). RSV and V29 promoted progression of the cell cycle of eBMMSCs into the S phase and may be useful for eBMMSC expansion.
The interfascicular matrix (IFM) binds tendon fascicles and contains a population of morphologically distinct cells. However, the role of IFM-localised cell populations in tendon repair remains to be ...determined. The basement membrane protein laminin-α4 also localises to the IFM. Laminin-α4 is a ligand for several cell surface receptors, including CD146, a marker of pericyte and progenitor cells. We used a needle injury model in the rat Achilles tendon to test the hypothesis that the IFM is a niche for CD146+ cells that are mobilised in response to tendon damage. We also aimed to establish how expression patterns of circulating non-coding RNAs alter with tendon injury and identify potential RNA-based markers of tendon disease. The results demonstrate the formation of a focal lesion at the injury site, which increased in size and cellularity for up to 21 days post injury. In healthy tendon, CD146+ cells localised to the IFM, compared with injury, where CD146+ cells migrated towards the lesion at days 4 and 7, and populated the lesion 21 days post injury. This was accompanied by increased laminin-α4, suggesting that laminin-α4 facilitates CD146+ cell recruitment at injury sites. We also identified a panel of circulating microRNAs that are dysregulated with tendon injury. We propose that the IFM cell niche mediates the intrinsic response to injury, whereby an injury stimulus induces CD146+ cell migration. Further work is required to fully characterise CD146+ subpopulations within the IFM and establish their precise roles during tendon healing.
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