Tendons and ligaments (T/Ls) play key roles in the musculoskeletal system, but they are susceptible to traumatic or age‐related rupture, leading to severe morbidity as well as increased susceptibility to degenerative joint diseases such as osteoarthritis. Tissue engineering represents an attractive therapeutic approach to treating T/L injury but it is hampered by our poor understanding of the defining characteristics of the two tissues. The present study aimed to determine differences in the proteomic profile between native T/Ls and tissue engineered (TE) T/L constructs. The canine long digital extensor tendon and anterior cruciate ligament were analyzed along with 3D TE fibrin‐based constructs created from their cells. Native tendon and ligament differed in their content of key structural proteins, with the ligament being more abundant in fibrocartilaginous proteins. 3D T/L TE constructs contained less extracellular matrix (ECM) proteins and had a greater proportion of cellular‐associated proteins than native tissue, corresponding to their low collagen and high DNA content. Constructs were able to recapitulate native T/L tissue characteristics particularly with regard to ECM proteins. However, 3D T/L TE constructs had similar ECM and cellular protein compositions indicating that cell source may not be an important factor for T/L tissue engineering.

Tendons and ligaments play key roles in the musculoskeletal system in both man and animals. Both tissues can undergo traumatic injury, age‐related degeneration and chronic disease, causing discomfort, pain and increased susceptibility to wider degenerative joint disease. To date, tendon and ligament ultrastructural biology is relatively under‐studied in healthy, non‐diseased tissues. This information is essential to understand the pathology of these tissues with regard to function‐related injury and to assist with the future development of tissue‐engineered tendon and ligament structures. This study investigated the morphological, compositional and extracellular matrix protein distribution differences between tendons and ligaments around the non‐diseased canine stifle joint. The morphological, structural characteristics of different regions of the periarticular tendons and ligaments (the intra‐articular anterior cruciate ligament, the extra‐articular medial collateral ligament, the positional long digital extensor tendon and energy‐storing superficial digital flexor tendons) were identified using a novel semi‐objective histological scoring analysis and by determining their biochemical composition. Protein distribution of extracellular matrix collagens, proteoglycans and elastic fibre proteins in anterior cruciate ligament and long digital extensor tendon were also determined using immunostaining techniques. The anterior cruciate ligament was found to have significant morphological differences in comparison with the other three tissues, including less compact collagen architecture, differences in cell nuclei phenotype and increased glycosaminoglycan and elastin content. Intra‐ and interobserver differences of histology scoring resulted in an average score 0.7, indicative of good agreement between observers. Statistically significant differences were also found in the extracellular matrix composition in terms of glycosaminoglycan and elastin content, being more prominent in the anterior cruciate ligament than in the other three tissues. A different distribution of several extracellular matrix proteins was also found between long digital extensor tendon and anterior cruciate ligament, with a significantly increased immunostaining of aggrecan and versican in the anterior cruciate ligament. These findings directly relate to the different functions of tendon and ligament and indicate that the intra‐articular anterior cruciate ligament is subjected to more compressive forces, reflecting an adaptive response to normal or increased loads and resulting in different extracellular matrix composition and arrangement to protect the tissue from damage.

Tendons play a critical role in the transmission of forces between muscles and bones, and chronic tendon injuries and diseases are among the leading causes of musculoskeletal disability. Little is known about sex-based differences in tendon structure and function. Our objective was to evaluate the mechanical properties, biochemical composition, transcriptome, and cellular activity of plantarflexor tendons from four month old male and female C57BL/6 mice using in vitro biomechanics, mass spectrometry-based proteomics, genome-wide expression profiling, and cell culture techniques. While the Achilles tendons of male mice were approximately 6% larger than female mice (P<0.05), the cell density of female mice was around 19% larger than males (P<0.05). No significant differences in mechanical properties (P>0.05) of plantaris tendons were observed. Mass spectrometry proteomics analysis revealed no significant difference between sexes in the abundance of major extracellular matrix (ECM) proteins such as collagen types I (P=0.30) and III (P=0.68), but female mice had approximately two-fold elevations (P<0.05) in less abundant ECM proteins such as fibronectin, periostin, and tenascin C. The transcriptome of male and female tendons differed by only 1%. In vitro, neither the sex of the serum that fibroblasts were cultured in, nor the sex of the ECM in which they were embedded, had profound effects on the expression of collagen and cell proliferation genes. Our results indicate that while male mice expectedly had larger tendons, male and female tendons have very similar mechanical properties and biochemical composition, with small increases in some ECM proteins and proteoglycans evident in female tendons.

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Exploring the tendon proteome is a challenging but important task for understanding the mechanisms of physiological/pathological processes during ageing and disease and for the development of new treatments. Several extraction methods have been utilised for tendon mass spectrometry, however different extraction methods have not been simultaneously compared. In the present study we compared protein extraction in tendon with two chaotropic agents, guanidine hydrochloride (GnHCl) and urea, a detergent, RapiGest™, and their combinations for shotgun mass spectrometry. An initial proteomic analysis was performed following urea, GnHCl, and RapiGest™ extraction of equine superficial digital flexor tendon (SDFT) tissue. Subsequently, another proteomic analysis was performed following extraction with GnHCl, Rapigest™, and their combinations. Between the two chaotropic agents, GnHCl extracted more proteins, whilst a greater number of proteins were solely identified after Rapigest™ extraction. Protein extraction with a combination of GnHCl followed by RapiGest™ on the insoluble pellet demonstrated, after label‐free quantification, increased abundance of identified collagen proteins and low sample to sample variability. In contrast, GnHCl extraction on its own showed increased abundance of identified proteoglycans and cellular proteins. Therefore, the selection of protein extraction method for tendon tissue for mass spectrometry analysis should reflect the focus of the study.

Background Osteoarthritis remains one of the greatest causes of morbidity and mortality in the equine population. The inability to detect pre-clinical changes in osteoarthritis has been a significant impediment to the development of effective therapies against this disease. Synovial fluid represents a potential source of disease-specific small non-coding RNAs (sncRNAs) that could aid in the understanding of the pathogenesis of osteoarthritis. We hypothesised that early stages of osteoarthritis would alter the expression of sncRNAs, facilitating the understanding of the underlying pathogenesis and potentially provide early biomarkers. Methods Small RNA sequencing was performed using synovial fluid from the metacarpophalangeal joints of both control and early osteoarthritic horses. A group of differentially expressed sncRNAs was selected for further validation through qRT-PCR using an independent cohort of synovial fluid samples from control and early osteoarthritic horses. Bioinformatic analysis was performed in order to identify putative targets of the differentially expressed microRNAs and to explore potential associations with specific biological processes. Results Results revealed 22 differentially expressed sncRNAs including 13 microRNAs; miR-10a, miR-223, let7a, miR-99a, miR-23b, miR-378, miR-143 (and six novel microRNAs), four small nuclear RNAs; U2, U5, U11, U12, three small nucleolar RNAs; U13, snoR38, snord96, and one small cajal body-specific RNA; scarna3. Five sncRNAs were validated; miR-223 was significantly reduced in early osteoarthritis and miR-23b, let-7a-2, snord96A and snord13 were significantly upregulated. Significant cellular actions deduced by the differentially expressed microRNAs included apoptosis (P < 0.0003), necrosis (P < 0.0009), autophagy (P < 0.0007) and inflammation (P < 0.00001). A conservatively filtered list of 57 messenger RNA targets was obtained; the top biological processes associated were regulation of cell population proliferation (P < 0.000001), cellular response to chemical stimulus (P < 0.000001) and cell surface receptor signalling pathway (P < 0.000001). Conclusions Synovial fluid sncRNAs may be used as molecular biomarkers for early disease in equine osteoarthritic joints. The biological processes they regulate may play an important role in understanding early osteoarthritis pathogenesis. Characterising these dynamic molecular changes could provide novel insights on the process and mechanism of early osteoarthritis development and is critical for the development of new therapeutic approaches.

BackgroundOsteoarthritis remains one of the greatest causes of morbidity and mortality in the equine population. The inability to detect pre-clinical changes in osteoarthritis has been a significant impediment to the development of effective therapies against this disease. Synovial fluid represents a potential source of disease-specific small non-coding RNAs (sncRNAs) that could aid in the understanding of the pathogenesis of osteoarthritis. We hypothesised that early stages of osteoarthritis would alter the expression of sncRNAs, facilitating the understanding of the underlying pathogenesis and potentially provide early biomarkers.MethodsSmall RNA sequencing was performed using synovial fluid from the metacarpophalangeal joints of both control and early osteoarthritic non-Thoroughbred horses. A group of differentially expressed sncRNAs was selected for further validation through qRT-PCR using an independent cohort of synovial fluid samples from control and early osteoarthritic horses. Bioinformatic analysis was performed in order to identify putative targets of the differentially expressed microRNAs and to explore potential associations with specific biological processes.ResultsResults revealed 22 differentially expressed sncRNAs including 13 microRNAs; miR-10a, miR-223, let7a, miR-99a, miR-23b, miR-378, miR-143 (and six novel microRNAs), four small nuclear RNAs; U2, U5, U11, U12, three small nucleolar RNAs; U13, snoR38, snord96, and one small cajal body-specific RNA; scarna3. Five sncRNAs were validated; miR-223 was significantly reduced in early OA and miR-23b, let-7a-2, snord96A and snord13 were significantly upregulated. Significant cellular functions deduced by the differentially expressed microRNAs included apoptosis (P < 0.0003), necrosis (P < 0.0009), autophagy (P < 0.0007) and inflammation (P < 0.00001). A conservatively filtered list of 57 messenger RNA targets was obtained; the top biological processes associated were regulation of cell population proliferation (P < 0.000001), cellular response to chemical stimulus (P < 0.000001) and cell surface receptor signalling pathway (P < 0.000001).ConclusionsSynovial fluid sncRNAs can be used as molecular biomarkers for early disease in equine osteoarthritic joints. The biological processes they regulate may play an important role in understanding early osteoarthritis pathogenesis. Characterising these dynamic molecular changes could provide novel insights on the process and mechanism of early osteoarthritis development and is critical for the development of new therapeutic approaches.

Ageing is a leading risk factor predisposing cartilage to osteoarthritis. However, little research has been conducted on the effect of ageing on the expression of small non-coding RNAs (sncRNAs). RNA from young and old chondrocytes from macroscopically normal equine metacarpophalangeal joints was extracted and subjected to small RNA sequencing (RNA-seq). Differential expression analysis was performed in R using package DESeq2. For transfer RNA (tRNA) fragment analysis, tRNA reads were aligned to horse tRNA sequences using Bowtie2 version 2.2.5. Selected microRNA (miRNAs or miRs) and small nucleolar RNA (snoRNA) findings were validated using real-time quantitative Polymerase Chain Reaction (qRT-PCR) in an extended cohort of equine chondrocytes. tRNA fragments were further investigated in low- and high-grade OA human cartilage tissue. In total, 83 sncRNAs were differentially expressed between young and old equine chondrocytes, including miRNAs, snoRNAs, small nuclear RNAs (snRNAs), and tRNAs. qRT-PCR analysis confirmed findings. tRNA fragment analysis revealed that tRNA halves (tiRNAs), tiRNA-5035-GluCTC and tiRNA-5031-GluCTC-1 were reduced in both high grade OA human cartilage and old equine chondrocytes. For the first time, we have measured the effect of ageing on the expression of sncRNAs in equine chondrocytes. Changes were detected in a number of different sncRNA species. This study supports a role for sncRNAs in ageing cartilage and their potential involvement in age-related cartilage diseases.