Objectives. The goal of this study was to determine whether intra-articular administration of the potentially anti-fibrotic agent decorin influences the expression of genes involved in the fibrotic cascade, and ultimately leads to less contracture, in an animal model. Methods. A total of 18 rabbits underwent an operation on their right knees to form contractures. Six limbs in group 1 received four intra-articular injections of decorin; six limbs in group 2 received four intra-articular injections of bovine serum albumin (BSA) over eight days; six limbs in group 3 received no injections. The contracted limbs of rabbits in group 1 were biomechanically and genetically compared with the contracted limbs of rabbits in groups 2 and 3, with the use of a calibrated joint measuring device and custom microarray, respectively. Results. There was no statistical difference in the flexion contracture angles between those limbs that received intra-articular decorin versus those that received intra-articular BSA (66° vs 69°; p = 0.41). Likewise, there was no statistical difference between those limbs that received intra-articular decorin versus those who had no injection (66° vs 72°; p = 0.27). When compared with BSA, decorin led to a statistically significant increase in the mRNA expression of 12 genes (p < 0.01). In addition, there was a statistical change in the mRNA expression of three genes, when compared with those without injection. . Conclusions. In this model, when administered intra-articularly at eight weeks, 2 mg of decorin had no significant effect on joint contractures. However, our genetic analysis revealed a significant alteration in several fibrotic genes. Cite this article: Bone Joint Res 2014;3:82–8

Summary. Based upon genetic analysis, decorin is an exciting pharmacologic agent of potential anti-fibrogenic effect on arthrofibrosis in our animal model. Introduction. While the pathophysiology of arthrofibrosis is not fully understood, some anti-fibrotic molecules such as decorin could potentially be used for the prevention or treatment of joint stiffness. The goal of this study was to determine whether intra-articular administration of decorin influences the expression of genes involved in the fibrotic cascade ultimately leading to less contracture in an animal model. Material and Methods. Eighteen rabbits had their right knees operated on to form contractures. The left knees served as controls. The 6 right limbs in the experimental group (Group 1) received four 500 ug/ml intra-articular injections of decorin over 8 days starting at 8 week, for a total of 2 mg. The 6 right limbs in the first control group (Group 2) received four intra-articular injections of bovine serum albumin (BSA) over 8 days starting at 8 weeks as well. The 6 six right limbs in the second control group (Group 3) received no injections. The contracted limbs of rabbits in Group 1 were biomechanically and genetically compared to the contracted limbs of rabbits in Groups 2 and 3 with the use of a calibrated joint measuring device and custom microarray, respectively. Results. There was no statistical difference in the flexion contracture angles between those right limbs that received intra-articular decorin versus those that received intra-articular BSA (66° vs. 69°; p = 0.41). Likewise, there was no statistical difference between those right limbs that received intra-articular decorin as opposed to those who had no injection (66° vs. 72°; p = 0.27). The lack of significance remained when the control left limbs were taken into account (p > 0.40). When compared to bovine serum albumin (BSA), decorin led to a statistically significant increase in the mRNA expression of 5 genes: substance P, neuropeptide γ, and neurokinin A, cyclin E2, and MMP-9 (p < 0.001). In addition, there was a statistically significant decrease in fibroblast growth factor receptor-2 (FGFR-2), rho-associated coiled-coil containing protein kinase-1 (ROCK-1), and vascular cell adhesion molecule-1 (VCAM-1) genes when intra-articular decorin was compared to no injection (p < 0.001). Conclusions. In this model, when administered intra-articularly at 8 weeks, 2 mg of decorin had no significant effect on joint contractures. However, our genetic analysis revealed a significant alteration in the expression of several fibrotic genes. Further studies investigating the route of administration, dosing, frequency, and timing are required before definitive conclusions may be drawn on the effects of decorin on joint contractures

Objectives. The aims of this study were to determine whether the administration of anti-inflammatory and antifibrotic agents affect the proliferation, viability, and expression of markers involved in the fibrotic development of the fibroblasts obtained from arthrofibrotic tissue in vitro, and to evaluate the effect of the agents on arthrofibrosis prevention in vivo. Methods. Dexamethasone, diclofenac, and decorin, in different concentrations, were employed to treat fibroblasts from arthrofibrotic tissue (AFib). Cell proliferation was measured by DNA quantitation, and viability was analyzed by Live/Dead staining. The levels of procollagen type I N-terminal propeptide (PINP) and procollagen type III N-terminal propeptide (PIIINP) were evaluated with enzyme-linked immunosorbent assay (ELISA) kits. In addition, the expressions of fibrotic markers were detected by real-time polymerase chain reaction (PCR). Fibroblasts isolated from healthy tissue (Fib) served as control. Further, a rabbit model of joint contracture was used to evaluate the antifibrotic effect of the three different agents. Results. Dexamethasone maintained the viability and promoted the proliferation of AFib. Diclofenac decreased the viability and inhibited the cell proliferation during the first week of cultivation. However, decorin inhibited AFib proliferation and downregulated the expressions of fibrotic markers. Additionally, decorin could improve the flexion contracture angle and inhibit the deposition of interstitial matrix components in the rabbit joint model. Conclusion. Decorin decreased the expression of myofibroblast markers in AFib, inhibited the proliferation of AFib, and prevented the initial procedure of arthrofibrosis in vivo, suggesting that decorin could be a promising treatment to inhibit the development of arthrofibrosis. Cite this article: X. Tang, S. Teng, M. Petri, C. Krettek, C. Liu, M. Jagodzinski. The effect of anti-inflammatory and antifibrotic agents on fibroblasts obtained from arthrofibrotic tissue: An in vitro and in vivo study. Bone Joint Res 2018;7:213–222. DOI: 10.1302/2046-3758.73.BJR-2017-0219.R2

Injured skeletal muscle repairs spontaneously via regeneration, however, this process is often incomplete because of fibrotic tissue formation. In our study we wanted to show improved efficiency of regeneration process induced by antifibrotic agent decorin in a combination with Platelet Rich Plasma (PRP)-derived growth factors. A novel human myoblast cell (hMC) culture, defined as CD56 (NCAM)+ developed in our laboratory, was used for evaluation of potential bioactivity of PRP and decorin. To determine the their effect on the viability of hMC we performed a MTT assay. To perform the cell proliferation assay, hMCs were separately seeded on plates at a concentration of 30 viable cells per well. Cell growth medium prepared with different concentrations of PRP exudates (5%, 10%, and 20%) and decorin (10 ng/mL, 25 ng/mL, and 50 ng/mL) were added and incubated for 7 days. After incubation we stained the cells with crystal-violet and measured the absorbance. To study the expression of Transforming Growth Factor Beta (TGF-β) and myostatin (MSTN), two main fibrotic factors in the process of muscle regeneration we performed several ELISA assays in groups treated with all therapeutic agents (PRP, decorin and their combination). Further, we have studied the ability of these agents to influence the differential cascade of dormant myoblasts towards fully differentiated myotubes by monitoring step wise activation of single nuclear factors like MyoD and Myogenin via multicolor flow cytometry. We stained the cells simultaneously with antibodies against CD56, MyoD and myogenin. We acquired cell images of 5,000 events per sample at 40 x magnification using 488 nm and 658 nm lasers and fluorescence was collected using three spectral detection channels. We analysed the cells populations according to expression of single or multiple markers and their ratios. Finally, we examined the treated cell populations using a multicolour laser microscope after staining for desmin (a key marker of myogenic differentiation of hMC), α-tubulin, and nuclei. Optical images were acquired at the center of chamber slides where the cell density is at its highest using a Leica TCS SP5 II confocal microscope and analysed using Photoshop CS6, where a “Color Range” tool was used in combination with a histogram palette to count the pixels that correspond to desmin-positive areas in an image. The mitochondrial activity of cells, as determined by the MTT assay, was significantly increased (p < 0 .001) after exposure to tested concentrations of PRP exudate. Similarly, viability was elevated in all tested concentrations of decorin. PRP exudate enhanced the viability of cells to more than 400% when compared to the control (p < 0 .001). The viability of cells treated with PRP exudates was also significantly higher when compared to decorin (p < 0 .001). Decorin did not show a significant effect on cell proliferation compared to the control, however, cultivation with PRP exudate leads to a 5-fold increase in cell proliferation (p < 0 .001). Decorin was shown to down-regulate the expression of TGF-β when compared to the control by more than 15% (p < 0 .001) but significantly less than PRP exudate p < 0 .005). PRP significantly down-regulated TGF-β expression by more than 30% (p < 0 .001). Similarly, the MSTN expression levels were significantly down-regulated by decorin and PRP. MSTN levels of cells treated with decorin were decreased by 28.4% (p < 0 .001) and 23.1% by PRP (p < 0 .001) when compared to the control group. Using flow cytometry we detected a 39.1% increase in count of myogenin positive cells in the PRP-treated group compared to the control. Moreover, there was a 3.09% increase in cells positive only for myogenin, whereas no such cells were found in the control cell population. The population of cells positive only for myogenin is considered as fully differentiated and capable of fusion into myotubes as well as future mucle fibers and is thus of great importance for muscle regeneration. At the same time 20.6% fewer cells remained quiescent (positive only for CD56). Cells positive for both MyoD and myogenin represent the population that shifted significantly towards mature myocites during myogenesis but are not yet fully committed. Finally, a statistically significant up-regulation of desmin expression (p < 0 .01 for the PRP treated group, p < 0 .005 for the decorin and PRP + decorin treated groups) was present in all therapeutic groups when compared to the control. While no significant difference was found between the PRP and decorin-treated groups, their combination led to a more than 3-fold increase (p < 0 .005) of desmin expression when compared to single bioactives. PRP can be a highly potential therapeutic agent for skeletal muscle regeneration and repair, especially if in combination with a TGF-β antagonis decorin. Achieving better healing could likely result in faster return to play and lower reinjury rate

Intervertebral disc (IVD) degeneration occurs with aging, leading to low back pain (LBP), which is one of the leading conditions of disability worldwide. With the lack of effective treatment, decellularized extracellular matrix (dECM) – based biomaterials have been proposed for IVD regeneration. However, the impact of donor ages on tissue repair had never been explored before in the disc field. Therefore, we aimed to address this question. For that, a decellularization protocol for bovine nucleus pulposus (NP) of different aged donors (fetus, young and old) was optimized by testing several detergents (SDS and Triton). The process efficiency was evaluated in terms of DNA and cell removal, as well as ECM preservation. Afterwards, dECMs were repopulated with bovine NP cells and cultured ex vivo. At day 7, cell behavior, ECM de novo synthesis and remodeling were evaluated [1]. Moreover, dECMs’ inflammatory response was assessed after in vivo CAM assay. Finally, inflammatory and angiogenic cytokines were analyzed in the conditioned media-derived from dECMs by using a cytokine array. As results, an optimal decellularization protocol (SDS 0.1%, 1h), efficient at removing cells and DNA from bovine NPs, while preserving ECM cues of native tissues, was developed. After repopulation, aggrecan increased in younger NPs, while collagen 2 decreased which may be indicative of matrix remodeling [1]. After in vivo CAM assay, fetal dECMs showed the highest inflammatory response. Finally, no statistically significant changes of cytokines were detected in the matrices, despite for a trend of higher IFN-α, IFN-γ and LIF in fetal dECMs, IL-1β in young dECMs and Decorin in old dECMs. Overall, this work uncovered the importance of tissue donor ages for tissue regenerative purpose, opening new avenues for the development of appropriate therapeutic strategies for IVD degeneration. Acknowledgments: FCT, EUROSPINE, ON Foundation

Fragmentation of SLRPs, including decorin, biglycan, lumican, keratocan and fibromodulin, has been shown to occur in osteoarthritic articular cartilage. We have previously shown an increased expression of lumican and keratocan, in osteoarthritic articular cartilage. The long-term aim of this project is to develop ELISAs for the detection of SLRP metabolites, and validate these potential biomarkers with synovial fluid and serum samples from a large cohort of normal and osteoarthritic patients. Initially, we aimed to determine whether SLRPs could be detected in synovial fluid and whether they were post-translationally modified with glycosaminoglycan (GAG) attachments; and whether bovine nasal cartilage (BNC) would be a plentiful source of native SLRP for ELISA development. Proteoglycans were extracted from BNC in guanidine hydrochloride. BNC extract and bovine synovial fluid was separated on an associative CsCl gradient. BNC CsCl cuts containing sulphated GAG were further purified using anion exchange chromatography. SLRPs in each fraction were detected using Western Blotting. Human recombinant lumican was expressed in Chinese hamster ovary (CHO) cells. Monoclonal antibodies that recognise epitopes on the core protein of human and bovine lumican and decorin were purified from hybridoma media using Protein G and Protein A affinity chromatography respectively. Monoclonal antibody activity against native and recombinant SLRPs was then determined using a direct ELISA. Preliminary tests showed that bovine synovial fluid contains keratocan and lumican with GAG attachments. BNC is a good source of post-translationally modified decorin, keratocan and biglycan but lumican was present predominantly without GAG attachments. Human recombinant lumican was successfully expressed with GAG attachments by CHO cells. Initial tests showed that the mAb against decorin was able to detect native decorin, with GAG attachments, in direct ELISA conditions. We have identified a plentiful source of native SLRP and begun ELISA development to ascertain whether these proteoglycans are potential biomarkers of OA

One out of nine Canadian males would suffer prostate cancer (PC) during his lifetime. Life expectancy of males with PC has increased with modern therapy and 90% live >10 years. However, 20% of PC-affected males would develop incurable metastatic diseases. Bone metastases (BM) are present in ~80% of metastatic PC patients, and are the most severe complication of PC, generating severe pain, fractures, spinal cord compression, and death. Interestingly, PC-BMs are mostly osteoblastic. However, the structure of this newly formed bone and how it relates to pain and fracture are unknown. Due to androgen antagonist treatment, different PC phenotypes develop with differential dependency on androgen receptor (AR) signaling: androgen-dependent (AR+), double negative (AR-) and neuroendocrine. How these phenotypes are related to changes in bone structure has not been studied. Here we show a state-of-the-art structural characterization of PCBM and how PC phenotypes are associated to abnormal bone formation in PCBM. Cadaveric samples (n=14) obtained from metastases of PC in thoracic or lumbar vertebrae (mean age 74yo) were used to analyze bone structure. We used micro-computed tomography (mCT) to analyze the three-dimensional structure of the bone samples. After imaging, the samples were sectioned and one 3mm thick section was embedded in epoxy-resin, ground and polished. Scanning electron microscopy (SEM)/energy-dispersive X-ray spectroscopy (EDS) and quantitative backscattering electron (qBSE) imaging were used to determine mineral morphology and composition. Another section was used for histological analysis of the PC-affected bone. Collagen structure, fibril orientation and extracellular matrix composition were characterized using histochemistry. Additionally, we obtained biopsies of 3 PCBM patients undergoing emergency decompression surgery following vertebral fracture and used them for immunohistological characterization. By using mCT, we observed three dysmorphic bone patterns: osteolytic pattern with thinned trabecula of otherwise well-organized structures, osteoblastic pattern defined as accumulation of disorganized matrix deposited on pre-existing trabecula, and osteoblastic pattern with minimum residual trabecula and bone space dominated by accumulation of disorganized mineralized matrix. Comparing mCT data with patho/clinical parameters revealed a trend for higher bone density in males with larger PSA increase. Through histological sections, we observed that PC-affected bone, lacks collagen alignment structure, have a higher number of lacunae and increased amount of proteoglycans as decorin. Immunohistochemistry of biopsies revealed that PC-cells inside bone organize into two manners: i) glandular-like structures where cells maintain their polarization in the expression of prostate markers, ii) diffuse infiltrate that spreads along bone surfaces, with loss of cell polarity. These cells take direct contact with osteoblasts in the surface of trabecula. We define that PCBM are mostly composed by AR+ with some double negative cells. We did not observe neuroendocrine phenotype cells. PCBMs generate predominantly osteoblastic lesions that are characterized by high lacunar density, lack of collagen organization and elevated proteoglycan content. These structural changes are associated with the infiltration of PC cells that are mostly androgen-dependent but have lost their polarization and contact directly with osteoblasts, perhaps altering their function. These changes could be associated with lower mechanical properties that led to fracture and weakness of the PCBM affected bone

Introduction. Energy storing tendons such as the equine superficial digital flexor tendon (SDFT) stretch and recoil with each stride and therefore require a high degree of compliance compared to tendons with a purely positional function, such as the equine common digital extensor tendon (CDET). This extra extensibility is provided by a specialised interfascicular matrix (IFM), which provides greater sliding and recoil between adjacent fascicles in energy storing tendons. However, the composition of the IFM remains largely undefined. We hypothesised that the IFM in the SDFT has a distinct composition, with a greater abundance of proteoglycans and elastin which facilitate extension and recoil. Materials and Methods. Transverse and longitudinal sections were cut from the mid-metacarpal regions of SDFTs and CDETs from 5 horses aged 3–7 years. Sections were stained using Alcian blue/Periodic acid Schiff to detect proteoglycans, elastic Van Giesson's to detect elastin, and immunohistochemistry was performed using antibodies for decorin, biglycan, fibromodulin, lumican and lubricin. Resultant images were graded by blinded observers to assess staining intensity in the IFM and fascicular matrix (FM), and statistical significance determined using ANOVA. Results. Overall proteoglycan abundance was significantly greater in the SDFT than the CDET (p<0.0001). In the SDFT, overall proteoglycan staining was greater in the FM than the IFM (p<0.0001). Elastin content was greater in the SDFT, where it was predominantly localised to the IFM. Fibromodulin staining was significantly greater in the CDET than the SDFT (p<0.05), whereas decorin staining was greater in the SDFT (p<0.05). In the SDFT, lubricin and biglycan staining were significantly greater in the IFM than the FM (p<0.05). Lumican staining was significantly greater in the CDET IFM than in the SDFT IFM (p<0.01). Discussion. As hypothesised, the IFM has a specialised composition in the SDFT, with a greater abundance of elastin, lubricin and biglycan. It is likely that the greater abundance of lubricin facilitates sliding between fascicles, whereas the localisation of elastin to the IFM is likely to provide the superior ability to recoil. The differential abundance of decorin, fibromodulin and lumican across tendon regions and types may reflect the different roles of these proteoglycans in tendon

Background. Hyperlaxity is associated with a high incidence of shoulder dislocations. Collagen V regulates the diameter of fibrils of the abundant collagen type I. Decorin and biglycan are members of the small leucine rich proteoglycans(SLRP's)family and play important roles in the regulation of collagen fibrillogenesis. The aim of this study was to identify if there was a link in hyperlaxity, capsule strength, collagen V and SLRP's expression. Methods. Data was collected for 10 patients undergoing open shoulder stabilization for recurrent instability. Beighton score was used to assess hyperlaxity. Localization of Collagen V and SLRP's was studied by immunohistochemical staining of paraffin embedded sections of shoulder capsule. Grading of the stain was done on a 0-4 scale(0=no staining and 4=strong staining>50% of the slide)by three observers. Shoulder capsules were mounted on a material testing system and vertical load was applied to reach yield. Results. The mean force required for yield in 15 shoulder capsules was 45N(17-78). Data was analysed for Group A(weak group) with yield<45N(8 specimens) and Group B(strong group)with yield>45N(7 specimens). The mean age was 26 years and all were male. The mean force for group A was 31N(17-41) and group B was 59N(45-78). The mean Beighton score for group A was 1.9(0-4) and Group B was 2. 2 specimens in Group A had Beighton score>4 as compared to 0 in Group B, indicating hyperlaxity. The mean grading of collagen V expression in synovial surface was 2.6,Blood vessels(BV)1.6 and extracellular matrix(ECM)1.9 in Group A and 4,3.1 and 2.6 respectively in group B. The mean grading of decorin expression for shoulder capsule was 2.7 in Group A and 3.3 in group B. The mean grading of Biglycan expression in synovial surface was 2,BV 2 and ECM 2.9 in Group A and 2,2.5 and 4 respectively in group B. Conclusions. We found that weaker capsule specimen(group A)had higher incidence of hyperlaxity. Decorin and biglycan expression in ECM and Collagen V expression in synovial surface, BV and ECM of shoulder capsule was higher in group B(strong group). This study shows a link between hyperlaxity, strength, Collagen V and SLRP's expression in shoulder capsule

Objectives. This study aimed to investigate time-dependent gene expression of injured human anterior cruciate ligament (ACL), and to evaluate the histological changes of the ACL remnant in terms of cellular characterisation. Methods. Injured human ACL tissues were harvested from 105 patients undergoing primary ACL reconstruction and divided into four phases based on the period from injury to surgery. Phase I was < three weeks, phase II was three to eight weeks, phase III was eight to 20 weeks, and phase IV was ≥ 21 weeks. Gene expressions of these tissues were analysed in each phase by quantitative real-time polymerase chain reaction using selected markers (collagen types 1 and 3, biglycan, decorin, α-smooth muscle actin, IL-6, TGF-β1, MMP-1, MMP-2 and TIMP-1). Immunohistochemical staining was also performed using primary antibodies against CD68, CD55, Stat3 and phosphorylated-Stat3 (P-Stat3). . Results. Expression of IL-6 was mainly seen in phases I, II and III, collagen type 1 in phase II, MMP-1, 2 in phase III, and decorin, TGF-β1 and α-smooth muscle actin in phase IV. Histologically, degradation and scar formation were seen in the ACL remnant after phase III. The numbers of CD55 and P-Stat3 positive cells were elevated from phase II to phase III. . Conclusions. Elevated cell numbers including P-Stat3 positive cells were not related to collagens but to MMPs’ expressions

Cell sheets are manufactured from a high-density cell layer stabilized by its own freshly produced extracellular matrix (ECM). They could serve as versatile scaffolds for tissue repair. Unfortunately, their production often remains time-consuming requiring weeks of culturing. Ligament cell sheets are so far barely available. Regarding musculoskeletal tissues exposed to high repetitive biomechanical forces, the stability of cell sheets is insufficient. It could help to combine them with a biomechanical competent scaffold e.g. produced by an embroidering technique. Hence, we wanted to (1) develop a very rapid strategy to produce ACL ligamentocyte sheets within 24 h by using a thermoresponsive polymer surface, (2) use the sheets for scaffold seeding and (3) reflect the fibrocartilaginous transition zone of an ACL enthesis by combining sheets of ligamentocytes with chondrocytes or chondrogenic precursor cells as a strategy for directed seeding of two cell types on topologically different scaffold areas. Different cell numbers of lapine ACL ligamentocytes (L-ACLs), lapine articular chondrocytes (L-ACs) and human mesenchymal stromal cells (H-MSCs) were used for sheet formation. Experiments were performed with novel, self-assembled poly(glycidyl ether) (PGE) brushes based on random glycidyl methyl ether and ethyl glycidyl ether copolymers on polystyrene 12-well cell culture plates, which allow rapid sheet formation within 24 h. Uncoated plates served as controls. Temperature-triggered detachment was performed by 10 min incubation with PBS at ambient temperature before treatment with fresh warm PBS for 5 min at 37 degrees Celsius. Harvested cell sheets were transferred on polyglycolic acid (PGA) or embroidered poly-lactic acid / poly-co-caprolactone (PLA/P[LA-CL]) scaffolds, functionalized with collagen foam and fluorine gas treatment (prepared at the IPF in Dresden and the FILK in Freiberg). Cell distribution, growth, vitality and synthesis of ECM components were monitored up to 7 days. Cell numbers required for sheet preparation (3.9 cm2) depended strongly on the cell type (L-ACLs: 0.395 mio/cm2, L-AC: 0.342 mio/cm2, H-MSCs: 0.131 mio/cm2) and was highest for L-ACLs. The majority of cells survived sheet assembly, detachment, transfer onto the scaffolds and culturing. Cells migrated from the sheets into the scaffolds and spread through the scaffolds. L-ACLs and L-ACs produced ECM and maintained their phenotypes (type II collagen and sulfated glycosaminoglycans in L-AC sheets, decorin and tenascin C in L-ACL sheets). The presence and distribution of two cell types in scaffold cocultures (L-ACLs and H-MSCs) was proven by anti-human vimentin labeling. Hence, the PGE brush surface allows rapid formation (24 h) of cell sheets

Introduction. Tendons and ligaments (TLs) play key roles in the musculoskeletal system. However, they are commonly damaged due to age-related wear and tear or torn in traumatic/sport related incidents resulting in pain and immobility. TLs contain cells and extracellular matrix (ECM) comprised of collagen, elastin, glycoproteins and proteoglycans. Although TLs are composed of similar components, their precise composition and arrangement of matrix macromolecules differ to provide specific mechanical properties and functions. To date little is known about how the main ECM proteins are arranged between the two tissue types. This data will provide essential information on fundamental structure of TLs leading to increased understanding of the function relationship between these tissue types. The aim of this study was to compare tendon-ligament differences in their ECM distribution of collagens, proteoglycans and elastic fibres. Materials and Methods. Anterior cruciate ligament (ACL) and long digital extensor tendon (LDET) were harvested from disease free cadaveric canine knee joints (n=3). Distributions of the main ECM components were assessed on longitudinal sections of ACL and LDET mid-substance. Antibody staining were assessed for collagen type I, III, VI, agreccan, versican, decorin, biglycan, elastin, fibrillin 1 and fibrillin 2. Results. Marked staining of collagen type I was present at fascicular regions, but also present at the interfascicular matrix (IFM). Collagen type III was present at the IFM of tendon, whilst in the ligament it was more widespread being located at both fascicles and IFM. In both TLs, collagen type VI was localised at IFM, but also present surrounding TL fibroblasts. A marked staining of aggrecan and versican was observed in ligament IFM, with pericellular staining of aggrecan present only in ligament. Decorin was found in both fascicular and IFM, whilst biglycan was occasionally present pericelullarly and at IFM in tendon. A similar pattern of elastic fibre distribution was found in both TLs. Discussion. This study has revealed a different ECM distribution of collagen type III, aggrecan, versican in ligament than when compared to tendon. These finding may relate to different functions between TLs and indicate that ligament is subjected to more compressive forces, resulting in different macromolecular arrangements that protect the tissue from damage

The enthesis is a tissue interface between tendon and bone, essential for adequate force transmission and composed by four distinct zones, namely tendon, fibrocartilage, mineralized fibrocartilage and bone. Given the avascularity of the tendon and the gradual change in tissue architecture and cell phenotype, the enthesis original tissue is often not re-established after chronic injuries, resulting in scar formation. Conservative treatments and surgical approaches are still far from a functional regeneration, whilst tissue engineering based scaffolds have recently showed great potential. In this work, we hypothesised that collagen-based scaffolds that mimic the basic architecture of the enthesis, will be able to spatially direct stem cell differentiation, providing an in vitro platform to study enthesis regeneration. A three-layer sponge composed of a tendon-like layer (collagen type I), a fibrocartilage-like layer (collagen type II) and a bone-like layer (collagen type I and hydroxyapatite) was fabricated by an iterative layering freeze-drying technique. Scaffold pore size and structural continuity at the interfaces were assessed by SEM and μ-CT analysis. Bone-marrow derived stem cells (BMSCs) were seeded on the scaffold and cultured in basal and differentiation media (chondrogenic, tenogenic and osteogenic). At day 7 and 21 the scaffolds were stained with Alizarin Red and Alcian Blue; alkaline phosphatase activity (ALP) and calcium and glycosaminoglycans (GAGs) were quantified in order to evaluate BMSC differentiation towards osteogenic and chondrogenic lineage. The presence of collagen I, III, tenascin and decorin in the scaffolds was evaluated by immunofluorescence staining in order to evaluate tenogenic differentiation of BMSCs. Scaffolds with three distinct but interconnected layers of collagen type I, collagen type II and collagen type I + hydroxyapatite were fabricated, with pore sizes in the range of 100–200 μm. Increased ALP and calcium levels were detected in a localised manner within the bone-like layer when scaffolds were cultured in basal medium (p<0.025 vs the other 2 layers). Similarly, proteoglycans were detected specifically in the fibrocartilage-like layer when scaffolds were cultured in the chondrogenic differentiation medium (p<0.03 vs the other 2 layers). Increased expression of tenogenic markers was observed in the tendon-like layer of scaffolds cultured in tenogenic media (p<0.045 vs the other 2 layers). In conclusion, the different collagen composition of each layer was able to spatially direct BMSC differentiation in a localized manner within the scaffold. Ongoing work is evaluating the synergistic effect between growth factor functionalized within the fibrocartilage and tendon-like layers for improved BMSC differentiation. Overall, these scaffolds hold promising potential in developing novel and more efficient strategy towards enthesis regeneration

Background. Proteoglycans (PGs) have long been known to be important to the functioning of the intervertebral disc. The most common PG is aggrecan, but there are also small leucine-rich proteoglycans (SLRPs) which constitute only a small percentage of the total PGs. However, they have many important functions, including organising the collagen, protecting it from degradation and attracting growth factors to the disc. We have examined how the core proteins of these molecules vary in intervertebral discs from patients with different pathologies. Methods. Discs were obtained from patients with scoliosis (n=7, 19–53y), degenerative disc disease (DDD) (n=6, 35–51y) and herniations (n=5, 33–58y). Proteoglycans were extracted and the SLRPs (biglycan, decorin, fibromodulin, keratocan and lumican) were characterised via Western blotting following enzymatic digestion with chondroitinase ABC and keratanase. Results. At least some SLRPs were present in all the discs studied. In addition to the presence of intact SLRP core proteins there was evidence of fragmentation of all the core proteins but especially of biglycan, fibromodulin and keratocan. Biglycan and keratocan were present in the majority of samples with biglycan being highly fragmented in the majority and keratocan usually present as 2 molecular weight bands. Fibromodulin was present in all samples except for 1 scoliotic disc and usually showed a high degree of fragmentation. The intact core protein of lumican was detected in all samples and was only present as a fragment in one of the older scoliosis samples. Decorin was present in a few samples of which half showed fragmentation. Conclusion. Although the number of samples investigated so far is low, fragmentation of these SLRP molecules appears common in the pathological intervertebral disc. These findings are useful not only in helping unravel pathways of disc degeneration, but may also provide early biomarkers of the different pathologies. Conflicts of Interest. None. Source of Funding. None. Acknowledgements: MRC and AR UK for financial support of SR & SO

Introduction: Tendon ruptures are increasingly common, repair can be difficult and healing poorly understood. Tissue engineering approaches often require expansion of cell numbers to populate a construct, and maintenance of cell phenotoype is essential for tissue regeneration. Methods: In this study we characterise the phenotype of human Achilles tenocytes and assess how this is affected by passaging. Tenocytes, isolated from tendon samples from 6 patients receiving surgery for rupture of the Achilles tendon, were passaged 8 times. Proliferation rates and cell morphology were recorded at passages 1, 4 and 8. Total collagen, the ratio of collagen types I and III and decorin were used as indicators of matrix formation and expression of the integrin ‘alpha’1 subunit as a marker of cell-matrix interactions. Results: With increasing passage number, cells became more rounded, were more widely spaced at confluence and confluent cell density declined from 18700 /cm2 to 16100 /cm2 (P=0.009). No change to total cell layer collagen was observed but the ratio of type III to type I collagen increased from 0.60 at passage 1 to 0.89 at passage 8 (P< 0.001). Decorin expression significantly decreased with passage number, from 22.9 ± 3.1 ng/ng DNA at passage 1, to 9.1 ± 1.8 ng/ngDNA at passage 8 (P< 0.001). Integrin expression did not change. Conclusion: We conclude that the phenotype of tenocytes in culture rapidly drifts with progressive passage

Background. Hyperlaxity is associated with a high incidence of sporting injuries. Collagen V regulates the diameter of fibrils of the abundant collagen type I. Decorin and biglycan are members of the small leucine rich proteoglycans(SLRP's)family and play important roles in the regulation of collagen fibrillogenesis. The aim of this study was to identify if there was a link in hyperlaxity, tissue strength, collagen V and SLRP's expression. Patients and methods. Data was collected for 25 patients. 12 had open shoulder stabilization and 13 had primary ACL reconstruction. Beighton score was used to assess hyperlaxity. Localization of Collagen V and SLRP's was studied by immunohistochemical staining of the paraffin embedded sections of the skin. Grading of the stain was done on a 0-4 scale(0=no staining and 4=strong staining>50% of the slide)by three observers. Tissue specimens were mounted on a material testing system and vertical load was applied to reach yield. Results. The mean force required for yield in 43 tissue specimens was 70N(12-171). Data was analysed for Group A(weak group)with yield<70N(21 tissue specimens)and Group B(strong group)with yield>70N(22 specimens). The mean age was 27 years. The mean force for group A was 41N(12-67)and group B was 98N(70-171). The mean Beighton score for group A was 3.4(0-9)and Group B was 1.9(0-5). 9 specimens in Group A and 4 in Group B had Beighton score>4 indicating hyperlaxity. The mean grading of collagen V expression in skin dermal papilla was 2.4, appendages 2.2 and extracellular matrix(ECM)1.8 in group A and 1.3,1.8 and 1.7 respectively in Group B. The mean grading of decorin expression in skin was 3.2 in group A and 3.1 in Group B. The mean grading of biglycan expression in skin epidermis was 1.5, appendages 2.2, ECM in superficial dermis 1.5 and deep dermis 0.75 in group A and 1.75,2.1,1.5 and 0.5 respectively in Group B. Conclusion. We found that weaker tissue specimen had high incidence of hyperlaxity and increased grading of expression for Collagen V in the skin dermal papillae. No difference was found in SLRP's expression in skin in both groups. The study shows a link between hyperlaxity, tissue strength and Collagen V expression in skin dermal papillae

Complex pathophysiologies involve different signalling mechanisms, with a multitude of often interconnected potential therapeutic targets. Therefore, there is a need for the development of multi-compartment delivery vehicles for combinatorial and synergistic therapeutic approaches. In this study it was hypothesized that multi-compartment crosslinked collagen type I systems can deliver multiple bioactive agents in a controlled manner in an in vitro model condition of skin fibrosis. Multi-compartment collagen-based systems were made using solutions of dialyzed type I collagen mixed with 10× PBS, after which they were neutralised and crosslinked with 1 and 2.0 mM 4 arm-succinimidyl glutarate ester PEG (4 arm-PEG-SG), respectively, followed by incubation at 37ºC. The systems were characterised through swelling assessment, collagenase degradation assay and compression tests. The release of encapsulated drugs from the hydrogels was studied by ELISA and the effect of the delivered bioactive agents was assessed through imaging and quantification for fibrotic markers in an in vitro model. A pilot study using FITC-dextran proved that the inner compartment was capable of promoting a sustained release over a long period of time (7 days), which was further confirmed with drug release assays using a TNF-α antagonist and recombinant decorin, fitting the intended therapeutic release profile. Protein expression studies showed a decrease of endogenous collagen type I and α-smooth muscle actin expression (p<0.05) indicating amelioration of fibrosis. In summary, this indicates that this system is suitable for dual delivery of multiple bioactive agents, resulting in a controlled release in vitro and illustrating its potential in therapy

Electromechanical coupling (piezoelectricity) is present in all living beings and provides basis for sense, thoughts and mechanisms of tissue regeneration. Herein, we ventured to assess the influence of MMC in mesenchymal stem cell culture. In this study, we fabricated piezoelectric regenerative scaffolds to assess the role of electromechamical stimulation on tendon regeneration. Tendon cells were selectively stimulated in vitro by mechanical or electromechanical cues using non-piezoelectric or piezoelectric scaffolds and optimal mechanical loading (4% deformation at 0.5 Hz). This was followed up with an in vivo study to assess tendon regeneration in a rat Achilles tendon injury model. P(VDF-TrFE), scaffolds were observed to mimic the fibrous structure of tendon tissue (figure 1) and were capable of producing electrical charges up to 17 pC/N when mechanically loaded (figure 1. Genes associated with tendon specific markers (Col.I/Col III, Scx and Mkx) and mechanosensitive ion channels such as PIEZO1, TRAAK and TRPV1 were significantly upregulated (figure 2). The upregulated genes were validated with individual real time Q-PCR and bioinformatics revealed a possible regulated function. Those results were further validated in vivo. Protein expression of repaired tendons showed a correlation between increase in expression of tendon related proteins SCX, TNMD, Decorin and expression of ion channels KCNK2, TRAAK and TRPV1. Collectively, these data clearly illustrate that scaffolds made of PVDF-TrFE can produce electrical charges when mechanically loaded. Moreover, gene and protein analyses showed a positive regulation of tendon specific markers through activation mechanosensitive voltage-gated genes. For any figures or tables, please contact authors directly

Introduction: Tendons consist of longitudinally running parallel bundles of collagen with rows of tendon cells between them. The tendon cells are linked to one another via gap junctions. 1. and cytoskeletally associated adherens junctions. Recent in vitro studies indicate that the two types of gap junction present, made of connexin 32 and connexin 43, regulate tendon cell responses to mechanical load. 2. In view of the importance of cell-cell interactions in tendon cell behaviour, we describe the behaviour of tendon cells in a novel 3-dimensional culture system designed to allow cells to establish cell-cell contacts and deposit matrix in the absence of scaffolds, which would favour cell-substrate interactions, and without disturbance by medium changes. Methods: Tendon fibroblasts were isolated from chicken tendons by protease and collagenase digestion. 3. , grown to passage 3 in HEPES buffered DMEM/5% foetal calf serum/1% antibiotic/1% L-glutamine at 370C, in 5% CO2 in air. Cells were suspended at 3x107 cells/ ml and 1 ml placed in a Spectrapor DispoDialyzerTM tube (MW cutoff 300,000). This was then placed into a 60 ml centrifuge tube with 40 ml DMEM containing ascorbate (1mg/ml) on a roller. Medium in the large tube was changed every 2 days. At 24 hours, 7, 14 and 21 days the cell aggregates were fixed in 90% methanol (4°C), frozen on dry ice and cryosections cut at 10–15 μm. Sections were labelled by indirect immunofluorescence with monoclonal type I, II, and III collagen, actin, vimentin and decorin, connexins 32 and 43, vinculin, Pan cadherin and N-cadherin. Results: Cells in suspension culture formed elongated aggregates up to 3cm long. Immunolabels showed that at 7 days type I and III collagens were present, predominantly in the periphery. At 14 days the collagens were uniformly distributed throughout the aggregates and showed parallel longitudinal organisation. It is also clear from propidium iodide label that the cell nuclei have distinct areas of alignment. The aggregates labelled positively for actin stress fibres, N-cadherin, decorin and connexin 32. Type II collagen and connexin 43 showed no conclusive label. Discussion: The suspension cultures clearly show that tendon cells can form large scale, organised structures in this culture system, and are capable of assembling an organised extracellular matrix in the absence of a scaffold to support them. The structures formed were similar to tendons in their cell and matrix organization. The amount of collagen deposited by the cells increases over time. Therefore, tendon cells could be used in a tissue engineering context to form well organised tissue in the absence of scaffolds in suitable culture systems. The presence of connexin 32 gap junctions and absence of connexin 43 shows the cell aggregates favouring matrix synthesis pathways – in cell cultures connexin 32 junctions promote collagen sythesis whereas connexin 43 inhibits it. 2.

Introduction. The ability of tendons to withstand stress generally decreases with age, often resulting in increased tissue degeneration and decreased regeneration capacity. However, the underlying molecular and cellular mechanisms of tendon senescence remain poorly characterized. Therefore, the aim of the current study was to identify genes showing an age-dependent altered expression profile in tendons. Materials and Methods. A suppression-subtractive-hybridization (SSH) screen comparing cDNA libraries generated from Achilles tendons of mature-adult (3 months) and old (18 months) female C57BL/6 mice was conducted. Subsequently, the differential expression of the identified genes was validated by RT-qPCR and selected genes were then further analysed by immunohistochemistry and Western blot. To investigate age-related structural alterations in the collagenous extracellular matrix we applied SHG-microscopy and TEM. In vitro experiments with young and old tendon derived stem/progenitor cells (TDSCs) involved wounding assays, tendon-like constructs as well as collagen gel contraction assays. Results. Among 168 identified genes, several ECM genes showed a differential expression, including Col1a1, Col3a1, fibronectin, fibromodulin, thrombospondin-1, decorin, biglycan, lysyl oxidase, and Sparc. As evidenced by RT-qPCR the mRNA levels of these genes were down-regulated in old tendons and in old TDSCs. Additionally, protein content of SPARC and Lysyl oxidase was diminished in vitro in cellular extracts from old TDSCs. The impact of Sparc on tendon ageing was further analysed in young and old Sparc−/− C57BL/6 as well as in age-matched wildtype mice. Tendons of Sparc−/− mice are generally thinner and TEM revealed thinner collagen fibrils and a larger interfibrillar area. Further, TDSCs of old and Sparc−/− tendons formed thinner in vitro tendon constructs, showed a higher collagen gel contraction capacity, and display altered cell-ECM adhesion and cell migration properties when compared to young wildtype cells. Employing SHG-microscopy we further observed age-related changes in the collagenous structure of Achilles tendons. Discussion. The decreased expression of ECM proteins and modulators thereof in old tendons in combination with structural changes is potentially associated with an increased risk of tendon injury in the elderly, since structure and composition of the tendon are directly related to its function