Autologous mesenchymal stem cell (MSC) injection into naturally-occurring equine tendon injuries has been shown to be safe and efficacious and protocols inform translation of the technique into humans. Efficient transfer of cells from the laboratory into tissue requires well-validated transport and implantation techniques.
Cell viability in a range of media was determined over 72 hours and after injection through a 19G, 21G or 23G needle. Viability, proliferation and apoptosis were analysed using TrypanBlue, alamarBlue® and AnnexinV assays.
Cell viability was similar in all re-suspension media following 24 hour storage, however cell death was most rapid in bone marrow aspirate, platelet-rich plasma and serum after longer storage. Cryogenic media exhibited greatest viability regardless of storage time. Cell proliferation after 24 and 72 hour storage was similar for all media, except after 24 hours in serum wherein proliferation was enhanced. MSC tri-lineage differentiation and viability did not significantly change when extruded through 19G, 21G or 23G needles, but 21G and 23G needles significantly increased apoptotic cells compared to 19G and non-injected controls. All gauges induced a decrease in metabolic activity immediately post-injection but cells recovered by 2 hours.
These data indicate storage and injection influence viability and subsequent cell behaviour and provide recommendations for MSC therapy that implantation of cells should occur within 24 hours of recovery from culture, using larger needle bores.
Injuries to the superficial digital flexor tendon (SDFT) are an important cause of morbidity and mortality in equine athletes, but the healing response is poorly understood. One important drive for the healing of connective tissues is the inflammatory cascade, but the role of inflammation in tendinopathy has been contentious in the literature. This article reviews the processes involved in the healing of tendon injuries in natural disease and experimental models. The importance of inflammatory processes known to be active in tendon disease is discussed with particular focus on recent findings related specifically to the horse.
Whilst inflammation is necessary for debridement after injury, persistent inflammation is thought to drive fibrosis, a perceived adverse consequence of tendon healing. Therefore the ability to resolve inflammation by the resident cell populations in tendons at an appropriate time would be crucial for successful outcome. This review summarises new evidence for the importance of resolution of inflammation after tendon injury. Given that many anti-inflammatory drugs suppress both inflammatory and resolving components of the inflammatory response, prolonged use of these drugs may be contraindicated as a therapeutic approach. We propose that these findings have profound implications not only for current treatment strategies but also for the possibility of developing novel therapeutic approaches involving modulation of the inflammatory process.
Tendon; Injury; Tendinopathy; Pathogenesis; Inflammation; Resolution; COX, cyclooxygenase; ECM, extracellular matrix; FPR2/ALX, lipoxin A4 receptor; IFN-γ, interferon gamma; IL-1β, interleukin-1beta; LXA4, lipoxin A4; Mφ, macrophage; mPGES-1, microsomal prostaglandin E synthase-1; MMP, matrix metalloproteinase; NSAIDs, non-steroidal anti-inflammatory drugs; PGE2, prostaglandin E2; SDFT, superficial digital flexor tendon; TNF-α, tumour necrosis factor alpha
•Sperm are mature cell types that can be transduced by lentiviral vectors.•Lentiviral integration in sperm has been demonstrated.•Lentivirally transduced sperm is useful for the study of early development.
Spermatozoa and lentiviruses are two of nature’s most efficient gene delivery vehicles. Both can be genetically modified and used independently for the generation of transgenic animals or gene transfer/therapy of inherited disorders. Here we show that mature spermatozoa can be directly transduced with various pseudotyped lentiviral vectors and used in in vitro fertilisation studies. Lentiviral vectors encoding Green Fluorescent Protein (GFP) were shown to be efficiently processed and expressed in sperm. When these transduced sperm were used in in vitro fertilisation studies, GFP expression was observed in arising blastocysts. This simple technique of directly transducing spermatozoa has potential to be a powerful tool for the study of early and pre-implantation development and could be used as a technique in transgenic development and vertical viral transmission studies.
GFP, Green Fluorescent Protein; LTR, Long Terminal Repeat; PGK, Phosphoglycerate kinase promoter; EF-1, Elongation factor 1 alpha promoter; CMV, Cytomegalovirus promoter; UCOE, ubiquitous chromatin opening element promoter; VSV-g, vesicular stomatitis virus; 293T, Human embryonic kidney cells; 7-AAD, 7-Aminoactinomycin D; IVF, in vitro fertilisation; AZT, azidodeoxythimidine; Lentiviral vectors; Spermatozoa; Transduction; Development; Transgenics; In vitro fertilisation
Background: Tendon disease is characterized by extensive remodeling of the extracellular matrix.
Results: Novel COMP cleavage fragments were identified in both an in vitro inflammatory model and natural disease.
Conclusion: Inflammatory mediators drive distinct COMP fragmentation at different stages of tendon disease.
Significance: Novel COMP neo-terminal fragments provide opportunities for developing markers for tendon injury.
During inflammatory processes the extracellular matrix (ECM) is extensively remodeled, and many of the constituent components are released as proteolytically cleaved fragments. These degradative processes are better documented for inflammatory joint diseases than tendinopathy even though the pathogenesis has many similarities. The aims of this study were to investigate the proteomic composition of injured tendons during early and late disease stages to identify disease-specific cleavage patterns of the ECM protein cartilage oligomeric matrix protein (COMP). In addition to characterizing fragments released in naturally occurring disease, we hypothesized that stimulation of tendon explants with proinflammatory mediators in vitro would induce fragments of COMP analogous to natural disease. Therefore, normal tendon explants were stimulated with IL-1β and prostaglandin E2, and their effects on the release of COMP and its cleavage patterns were characterized. Analyses of injured tendons identified an altered proteomic composition of the ECM at all stages post injury, showing protein fragments that were specific to disease stage. IL-1β enhanced the proteolytic cleavage and release of COMP from tendon explants, whereas PGE2 had no catabolic effect. Of the cleavage fragments identified in early stage tendon disease, two fragments were generated by an IL-1-mediated mechanism. These fragments provide a platform for the development of neo-epitope assays specific to injury stage for tendon disease.
Extracellular Matrix; Inflammation; Mass Spectrometry (MS); Peptides; Proteomics; Tendon
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. 1X107 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.
The contribution of inflammation to the pathogenesis of tendinopathy and high prevalence of re-injury is not well established, although recent evidence suggests involvement of prostaglandins. We investigated the roles of prostaglandins and inflammation-resolving mediators in naturally occurring equine tendon injury with disease stage and age. Levels of prostaglandins E2 (PGE2), F2α (PGF2α), lipoxin A4 (LXA4) and its receptor FPR2/ALX were analysed in extracts of normal, sub-acute and chronic injured tendons. To assess whether potential changes were associated with altered PGE2 metabolism, microsomal prostaglandin E synthase-1 (mPGES-1), prostaglandin dehydrogenase (PGDH), COX-2 and EP4 receptor expression were investigated. The ability of tendons to resolve inflammation was determined by assessing FPR2/ALX expression in natural injury and IL-1β stimulated tendon explants.
Alterations in the profile of lipid mediators during sub-acute injury included low PGE2 and elevated LXA4 levels compared to normal and chronic injuries. In contrast, PGF2α levels remained unchanged and were three-fold lower than PGE2. The synthetic capacity of PGE2 as measured by the ratio of mPGES-1:PGDH was elevated in sub-acute injury, suggesting aberrations in tendon prostaglandin metabolism, whilst COX-2 and EP4 receptor were unchanged. Paradoxically low tendon PGE2 levels in early injury may be attributed to increased local clearance via PGDH or the class switching of lipid mediators from the prostaglandin to the lipoxin axis. PGE2 is therefore implicated in the development of tendon inflammation and its ensuing resolution. Whilst there was no relationship between age and tendon LXA4 levels, there was an age-associated decline in FPR2/ALX receptor expression with concurrent increased PGE2 levels in injury. Furthermore, uninjured tendon explants from younger (<10 years) but not older horses (≥10 years) treated with IL-1β responded by increasing FPR2/ALX suggesting aged individuals exhibit a reduced capacity to resolve inflammation via FPR2/ALX, which may present a potential mechanism for development of chronic tendinopathy and re-injury.
Macrophages (Mϕ) orchestrate inflammatory and reparatory processes in injured connective tissues but their role during different phases of tendon healing is not known. We investigated the contribution of different Mϕ subsets in an equine model of naturally occurring tendon injury. Post mortem tissues were harvested from normal (uninjured), sub-acute (3–6 weeks post injury) and chronically injured (>3 months post injury) superficial digital flexor tendons. To determine if inflammation was present in injured tendons, Mϕ sub-populations were quantified based on surface antigen expression of CD172a (pan Mϕ), CD14highCD206low (pro-inflammatory M1Mϕ), and CD206high (anti-inflammatory M2Mϕ) to assess potential polarised phenotypes. In addition, the Lipoxin A4 receptor (FPR2/ALX) was used as marker for resolving inflammation. Normal tendons were negative for both Mϕ and FPR2/ALX. In contrast, M1Mϕ predominated in sub-acute injury, whereas a potential phenotype-switch to M2Mϕ polarity was seen in chronic injury. Furthermore, FPR2/ALX expression by tenocytes was significantly upregulated in sub-acute but not chronic injury. Expression of the FPR2/ALX ligand Annexin A1 was also significantly increased in sub-acute and chronic injuries in contrast to low level expression in normal tendons. The combination of reduced FPR2/ALX expression and persistence of the M2Mϕ phenotype in chronic injury suggests a potential mechanism for incomplete resolution of inflammation after tendon injury. To investigate the effect of pro-inflammatory mediators on lipoxin A4 (LXA4) production and FPR2/ALX expression in vitro, normal tendon explants were stimulated with interleukin-1 beta and prostaglandin E2. Stimulation with either mediator induced LXA4 release and maximal upregulation of FPR2/ALX expression after 72 hours. Taken together, our data suggests that although tenocytes are capable of mounting a protective mechanism to counteract inflammatory stimuli, this appears to be of insufficient duration and magnitude in natural tendon injury, which may potentiate chronic inflammation and fibrotic repair, as indicated by the presence of M2Mϕ.
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.
Methods and Findings
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.
A hallmark of rheumatoid arthritis (RA) is invasion of the synovial pannus into cartilage and this step requires degradation of the collagen matrix. The aim of this study was to explore the role of one of the collagen-degrading matrix metalloproteinases (MMPs), membrane-type 1 MMP (MT1-MMP), in synovial pannus invasiveness.
Expression and localization of MT1-MMP in human RA pannus were investigated by Western blot analysis of primary synovial cells and immunohistochemistry of RA joints specimens. The functional role of MT1-MMP was analyzed by 3D collagen invasion assays and a cartilage invasion assay in the presence or absence of tissue inhibitor of metalloproteinase (TIMP)-1, TIMP-2, or GM6001. The effect of adenoviral expression of a dominant negative MT1-MMP construct lacking a catalytic domain was also examined.
MT1-MMP was highly expressed at the pannus-cartilage junction of RA joints. Freshly isolated rheumatoid synovial tissues and isolated RA synovial fibroblasts invaded into a 3D collagen matrix in an MT1-MMP-dependent manner. Invasion was blocked by TIMP-2 and GM6001, but not by TIMP-1. It was also inhibited by the over-expression of a dominant negative MT1-MMP which inhibits collagenolytic activity and proMMP-2 activation by MT1-MMP on the cell surface. Synovial fibroblasts also invaded into cartilage in an MT1-MMP-dependent manner. This process was further enhanced by removing aggrecan from the cartilage matrix.
MT1-MMP is an essential collagen-degrading proteinase during pannus invasion in human RA. Specific inhibition of MT1-MMP-dependent invasion may form a novel therapeutic strategy for RA.
MT1-MMP; synovial pannus; rheumatoid arthritis