Anterior cruciate ligament reconstruction (ACLR) using a semitendinosus (ST) autograft, with or without gracilis (GR), results in donor muscle atrophy and varied tendon regeneration. The effects of harvesting these muscles on muscle moment arm and torque generating capacity have not been well described. This study aimed to determine between-limb differences (ACLR vs uninjured contralateral) in muscle moment arm and torque generating capacity across a full range of hip and knee motions. A secondary analysis of magnetic resonance imaging was undertaken from 8 individuals with unilateral history of ST-GR ACLR with complete ST tendon regeneration. All hamstring muscles and ST tendons were manually segmented. Muscle length (cm), peak cross-sectional area (CSA) (cm. 2. ), and volume (cm. 3. ) were measured in ACLR and uninjured contralateral limbs. OpenSim was used to simulate and evaluate the mechanical consequences of changes in normalised moment arm (m) and torque generating capacity (N.m) between ACLR and uninjured contralateral limbs. Compared to uninjured contralateral limbs, regenerated ST tendon re-insertion varied proximal (+) (mean = 0.66cm, maximum = 3.44cm, minimum = −2.17cm, range = 5.61cm) and posterior (+) (mean = 0.38cm maximum = 0.71cm, minimum = 0.02cm, range = 0.69cm) locations relative to native anatomical positions. Compared to uninjured contralateral limbs, change in ST tendon insertion point in ACLR limbs resulted in 2.5% loss in peak moment arm and a 3.4% loss in peak torque generating capacity. Accounting for changes to both max isometric force and ST moment arm, the ST had a 14.8% loss in peak torque generating capacity. There are significant deficits in ST muscle morphology and insertion points following ST-GR ACLR. The ST atrophy and insertion point migration following ACLR may affect force transmission and distribution within the hamstrings and contribute to persistent deficits in knee flexor and internal rotator strength

Adipose-derived stem cells (ADSCs) are an effective alternative for Teno-regeneration. Despite their applications in tendon engineering, the mechanisms promoting tendon healing still need to be understood. Since there is scattered information on ovine ADSCs, this research aims to investigate in vitro their teno-differentiation for potential use in preclinical tendon regeneration models. Ovine ADSCs were isolated from the tail region according to FAT-STEM laboratories, expanded until passage six (P6), and characterized in terms of stemness, adhesion and MHC markers by Flow Cytometry (FCM) and immunocytochemistry (ICC). Cell proliferation and senescence were evaluated with MTT and Beta-galactosidase assays, respectively. P1 ADSCs’ teno-differentiation was assessed by culturing them with teno-inductive Conditioned Media (CM) or engineering them on tendon-mimetic PLGA scaffolds. ADSCs teno-differentiation was evaluated by morphological, molecular (qRT-PCR), and biochemical (WesternBlot) approaches. ADSCs exhibited mesenchymal phenotype, positive for stemness (SOX2, NANOG, OCT4), adhesion (CD29, CD44, CD90, CD166) and MHC-I markers, while negative for hematopoietic (CD31, CD45) and MHC-II markers, showing no difference between passages. ICC staining confirmed these results, where ADSCs showed nuclear positivity for SOX2 (≅ 56%) and NANOG (≅ 67%), with high proliferation capacity without senescence until P6. Interestingly, ADSCs cultured with the teno-inductive CM did not express tenomodulin (TNMD) protein or gene. Conversely, ADSCs seeded on scaffolds teno-differentiated, acquiring a spindle shape supported by TNMD protein expression at 48h (p<0.05 vs. ADSCs 48h) with a significant increase at 14 days of culture (p<0.05 vs. ADSCs + fleece 48h). Ovine ADSCs respond differently upon distinct teno-inductive strategies. While the molecules on the CM could not trigger a teno-differentiation in the cells, the scaffold's topological stimulus did, resulting in the best strategy to apply. More insights are requested to better understand ovine ADSCs’ tenogenic commitment before using them in vivo for tendon regeneration. Acknowledgements: This research is part of the P4FIT project ESR5, under the H2020MSCA-ITN-EJD-P4 FIT-Grant Agreement ID:955685

Traumatic acute or chronic tendon injuries are a wide clinical problem in modern society, resulting in important economic burden to the health system and poor quality of life in patients. Due to the low cellularity and vascularity of tendon tissue the repair process is slow and inefficient, resulting in mechanically, structurally, and functionally inferior tissue. Tissue engineering and regenerative medicine are promising alternatives to the natural healing process for tendon repair, especially in the reconstruction of large damaged tissues. The aim of TRITONE project is to develop a smart, bioactive implantable 3D printed scaffold, able to reproduce the structural and functional properties of human tendon, using FDA approved materials and starting from MSC and their precursor, MPC cell mixtures from human donors. Total cohort selected in the last 12 months was divided in group 1 (N=20) of subjects with tendon injury and group 2 (N=20) of healthy subject. Groups were profiled and age and gender matched. Inclusion criteria were age>18 years and presence of informed consent. Ongoing pregnancy, antihypertensive treatment, cardiovascular diseases, ongoing treatment with anti-aggregants, acetylsalicylic-acid or lithium and age<18 years were exclusion criteria. Firstly, we defined clinical, biological, nutritional life style and genetic profile of the cohort. The deficiency of certain nutrients and sex hormonal differences were correlated with tendon-injured patients. It was established the optimal amount of MPC/MSC human cell (collected from different patients during femoral neck osteotomy). Finally, most suitable biomaterials for tendon regeneration and polymer tendon-like structure were identified. Hyaluronic acid, chemical surface and soft-molecular imprinting (SOFT-MI) was used to functionalize the scaffold. These preliminary results are promising. It will be necessary to enroll many more patients to identify genetic status connected with the onset of tendinopathy. The functional and structural characterization of smart bioactive tendon in dynamic environment will represent the next project step

Tendon injuries occur frequently in athletes and the general population, with inferior healing leading to deposition of fibrotic scar tissue. New treatments are essential to limit fibrosis and enable tendon regeneration post-injury. In this study, we tested the hypothesis that rapamycin improves tendon repair and limits fibrosis by inhibiting the mTOR pathway. The left hindlimb of female adult Wistar rats was injured by needle puncture and animals were either given daily injections of rapamycin (2mg/kg) or vehicle. Animals were euthanized 1 week or 3 weeks post-injury (n=6/group). Left and right Achilles tendons were harvested, with the right limbs acting as controls. Tendon sections were stained with haematoxylin & eosin, and scored by 2 blinded scorers, assessing alterations in cellularity, cell morphology, vascularity, extracellular matrix (ECM) organization and peritendinous fibrosis. Immunohistochemistry was performed for the tendon pan-vascular marker CD146 and the autophagy marker LC3. Injury resulted in significantly altered ECM organization, cell morphology and cellularity in both rapamycin and vehicle-treated groups, but no alterations in vascularity compared to uninjured tendons. Rapamycin had a limited effect on tendon repair, with a significant reduction in peritendinous fibrosis 3 weeks after injury (p=0.028) but no change in cell morphology, cellularity or ECM organization compared to vehicle treated tendons at either 1 week or 3 weeks post injury. CD146 labelling was increased at the site of injury, but there was no apparent difference in CD146 or LC3 labelling in rapamycin and vehicle treated tendons. The decrease in peritendinous fibrosis post-injury observed in rapamycin treated tendons indicates rapamycin as a potential therapy for tendon adhesions. However, the lack of improvement of other morphological parameters in response to rapamycin treatment indicates that rapamycin is not an effective therapy for injuries to the tendon core. Acknowledgements: This study was funded by Versus Arthritis (22607)

Tendon injuries are a common problem that can significantly impact an individual's quality of life. While traditional surgical methods have been used to address this issue, Extracellular Vesicles (EVs) have emerged as a promising approach to promote tendon repair and regeneration mechanisms, as they deliver specific biological signals to neighbouring cells. In this study, we extracted human Tendon Progenitor Stem cells (hTPSCs) from surgery explants and isolated their EVs from perfused and static media. hTPSCs were isolated from tendon surgery biopsy (Review Board prot./SCCE n.151, 29/10/2020) and cultured in both static and dynamic conditions, using a perfusion bioreactor (1ml/min). When cells reached 80% confluence, they were switched into a serum-free medium for 24 hours for EVs-production. Conditioned media was ultra-centrifuged for 90min (100000g). The recovered pellet was then characterized by size and concentration (Nanosight NS300), Zeta potential (Mastersizer S), morphology (SEM and TEM) and protein quantification. hTPSCs stemness and multipotency were confirmed through CD73, CD90, and CD105 expression and confirmation of quad-lineage (adipo-osteo-chondro-teno) differentiation. After 7 days, hTPSCs were ready for EVs-production. Ultracentrifugation revealed the presence of particles with a concentration of 7×107 particles/mL consistent across both cultures. Further characterization indicated that EVs collected from perfused conditions displayed an elevated vesicle mean size (mean 143±6.5 nm) in comparison to static conditions (mean 112±7.4 nm). Consistent with, but not in proportion with, the above protein content was measured at 20 ng/ml (dynamic) and 7 ng/mL (static) indicating a nearly 3-fold increase in concentration associated with a ~22% increase in particle size. Proposed data showed that sub-200 diameter vesicles were successfully collected from multipotent hTPSCs starvation, and the vesicle size and protein concentration were compatible with established EV literature; furthermore, dynamic culture conditions seemed more suitable for EVs-production. Further characterization will be required to better understand, EVs-compositions and their role in tendon regenerative events

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

The formation of postoperative adhesions poses a major complication in surgery, especially in the treatment of tendon, where adhesions can result in an alteration of the biomechanical and gliding properties, impeding a proper functioning of the tendon. Current treatments to prevent adhesions in the tendon are mainly based on the use of mechanical barriers which isolate the tendon and prevent fibrin deposition. Despite the positive results in preclinical models, these results have not been translated to clinics. Thus, in this study we propose a porcine peritoneum xenograft as an alternative antiadhesion barrier which integrates a basal membrane, since the presence of a basal membrane together with an epithelium or mesothelium layer prevents the formation of adhesions in vivo. First results have shown the suitability of the porcine peritoneum xenograft as an antiadhesion barrier due to its lower crosslinking ratio (p<0.05) and faster degradation by MMPs in vitro than a commercially available tendon product, which suggest a faster remodelling in vivo. On the other hand, the porcine peritoneum showed higher mechanical properties (p<0.01) and a lower coefficient of friction (p<0.01), characteristics that make the porcine peritoneum an appropriate material for tendon regeneration. Furthermore, the presence in the xenograft of a collagen type IV and laminin network after decellularisation was confirmed with immunohistochemistry, which poses the potential of the porcine peritoneum as antiadhesion device due to the presence of a basal membrane. Preliminary cell assessment experiments showed different morphology of adult dermal fibroblast (ADFs) on the different sides of the material (basal membrane and connective tissue) due to the differences in composition of both layers. Furthermore, the culture of ADFs during 7 days in media conditioned with the porcine peritoneum resulted in higher proliferation and metabolic activity (p<0.05) than those observed in the control and the media conditioned with the commercial product, suggesting the presence of growth factors in the porcine peritoneum which promote the growth of cells. Although positive results have been observed regarding the potential of porcine peritoneum as antiadhesion barrier for tendon regeneration, further studies which assess the influence of the basal membrane on cell behaviour and confirm the potential of the xenograft as antiadhesion barrier are being carried out

Tendon injuries are a worldwide problem affecting several age groups and stem cell based therapies hold potential for tendon strategies guiding tendon regeneration. Tendons rely on mechano-sensing mechanisms that regulate homeostasis and influence regeneration. The mechanosensitive receptors available in cell membranes sense the external stimuli and initiate mechanotransduction processes. Activins are members of the TGF-β superfamily which participate in several tendon biological processes. It is envisioned that the activation of the activin receptor, trigger downstream Smad2/3 pathway thus regulating the transcription of tenogenic genes driving stem cell differentiation. In this work, we propose to target the Activin receptor type IIA (ActRIIA) in human adipose stem cells (hASCs), inducing hASCs commitment towards the tenogenic lineage. Since mechanotransduction can be remotely triggered through magnetic actuation combined with magnetic nanoparticles (MNPs), we stimulated hASCs tagged complexes using a vertical oscillating magnetic bioreactor (MICA Biosystems Ltd). Carboxyl functionalised MNPs (Micromod) were coated with anti-ActRIIA antibody (Abcam) by carbodiimide activation. hASCs were then cultured with MNPs-anti-ActRIIA for 14days with or without magnetic exposure (1Hz, 1h/every other day). hASCs cultured alone in αMEM (negative control) or in αMEM supplemented with ActivinA (R&D systems) (positive control of ActRIIA activation) were used as experimental controls. The tenogenic commitment of hASCs was assessed by real time RT-PCR, immunocytochemistry and quantification of collagen and non-collagenous proteins. Moreover, the phosphorylation of Smad2/3 was also evaluated on hASCs incubated for 2, 10, or 30min under magnetic stimulated (1Hz) and non-stimulated conditions. The increased gene expression of tendon related markers and higher ECM proteins deposition suggests that remote magnetic activation of ActRIIA promotes effectively hASCs tenogenic commitment. Furthermore, the detection of phospho-Smad2/3 proteins by ELISA (Cell Signaling Technology) was significantly more intense after 10min in hASCs under magnetic stimulation and in comparison to the control groups. These outcomes suggest that ActRIIA is a mechanosensitive receptor that can be remotely activated upon magnetic stimulation. In conclusion, remotely activation of MNPs tagged hASCs has potential for modulating tenogenic differentiation of stem cells envisioning successful cell therapies for tendon regeneration. Acknowledgements. FCT/MCTES PD/59/2013 (fellowship PD/BD/113802/2015), FCT post-doctoral grant SFRH/BPD/111729/2015, FCT grant IF/00685/2012, and EU-ITN MagneticFun

Introduction. The rabbit common calcanean (Achilles) tendon is a compound apparatus frequently used in studies considering novel interventions to facilitate tendon regeneration. These studies often employ complete surgical transection of the apparatus. Due consideration of the translational relevance to human tendinopathy is often lacking and refinement of this injury model, consistent with the principles of the 3Rs, has not been forthcoming. Materials and Methods. Wild rabbit cadavers (n=10) were obtained from a licensed game dealer. For gross anatomy studies the caudal crus was dissected and transverse sections obtained every 5 mm. Ultrasongraphic examination of the entire apparatus was peformed with a 15 Hz transducer in transverse sections. Results. This study reannotates the apparatus and demonstrates that the principal structures, the superficial digital flexor tendon and medial and lateral gastrocnemius tendons, may be clearly identified by ultrasonographic examination. Discussion. Historical descriptions of the rabbit Achilles apparatus are shown to be inaccurate and follow human gross anatomical descriptions. Ultrasonographic identification of the constituent structures in the rabbit are poorly represented in the literature. Reference measurements and qualitative descriptions are provided that may facilitate the development of refined surgical techniques for in vivo studies of tendon regeneration in the rabbit beyond crude transection studies

Adherent cells are known to respond to physical characteristics of their surrounding microenvironment, adapting their cytoskeleton and initiating signaling cascades specific to the type of cue encountered. Scaffolds mimicking native biophysical cues have proven to differentiate stem cells towards tissue-specific lineages and to maintain the phenotype of somatic cells for longer periods of time in culture. Biomaterial-based tendon implants are designed to withstand high physiological loads but often lack the appropriate biochemical, biophysical and biological structure to drive tendon regeneration by populating cells. The objective of this study is to use tendon main component, collagen type I, to create scaffolds that reproduce tendon natural anisotropy and rigidity, in an effort to engineer functional tendon tissue with native organization and strength, able to maintain tenocyte phenotype and to differentiate stem cells towards the tenogenic lineage. Porcine collagen type I in solution was treated with one of the following cross-linkers: glutaraldehyde, genipin or 4-arm polyethylene glycol (4SP). The resulting mixture was poured on micro-grooved (2×2×2 um) or planar PDMS moulds and air-dried to obtain 5 mg/ml collagen films. Surface topography and elastic modulus were analyzed using SEM/AFM and rheometry, respectively. Human tendon cells were cultured on the micro-grooved/planar scaffolds for up to 10 days. Cell morphology, collagen III and tenascin C expression were analyzed by immunocytochemistry. Among the different cross-linkers used, only the treatment with 4SP resulted in scaffolds with a recognizable micro-grooved surface topography. Precise control over the micro-grooved topography and the rigidity of the scaffolds was achieved by cross-linking the collagen with varying concentrations of 4SP (0, 0.5, 1 and 1.5mM) at low pH and temperature. The elastic modulus of the scaffolds cross-linked with 4SP (0.5mM) matched the values previously reported to induce tenogenic differentiation in stem cells (50–90 kPa). Approximately eighty percent of the human tendon cells cultured on the micro-grooved collagen films aligned in the direction of the anisotropy for 10 days in culture, mimicking the alignment of tenocytes in the native tissue. Cell nuclei morphology, known to play a central role in the process of mechanotransduction, was significantly more elongated for the tenocytes cultured on the micro-grooved scaffolds after 4 days in culture for all the 4SP concentrations. Synthesis, deposition and alignment of collagen III and tenascin C, two important tenogenic markers, were up regulated selectively on the micro-grooved and rigid scaffolds after 10 days in culture, respectively. These results highlight the synergistic effect of matrix rigidity and cell alignment on tenogenic cell lineage commitment. Collectively, this study provides new insights into how collagen can be modulated to create scaffolds with precise imprinted topographies and controlled rigidities

Summary Statement. A resorbable and biocompatible polymer-based scaffold was used for the proliferation and delivery of adipose derived stromal cells, as well as delivery of a cell growth/differentiation promoting factor for improved tendon defect regeneration. Introduction. Surgeons perform thousands of direct tendon repairs annually. Repaired tendons fail to return to normal function following injury, and thus require continued efforts to improve patient outcomes. The ability to produce regenerate tendon tissue with properties equal to pre-injured tendon could lead to improved treatment outcomes. The aim of this study was to investigate in vivo tendon regeneration using a biodegradable polymer for the delivery of adipose derived stromal cells (ADSCs) and a polypeptide, growth/differentiation factor-5/(GDF-5), in a tendon gap model. Patients & Methods. Female Fischer 344 rats underwent unilateral Achilles tenotomies. Defects were left un-repaired (Group 1-control), bridged using electrospun 65:35 polylactide-co-glycolide (PLAGA) tubular scaffolds (Group 2), PLAGA/ADSCs (Group 3), or PLAGA/GDF-5 (Group 4) scaffold composites. The plantaris was left intact. Operative limbs were immobilised for 10–14 days, followed by unrestricted activity. The rats were sacrificed at 4 weeks or 8 weeks after surgery, and tendons were assessed with histological, biochemical, and mechanical analyses. Results. PLAGA, PLAGA/ADSCs, and PLAGA/GDF-5 groups showed increased collagen I gene expression at both the 4 and 8 week time points (p<0.05). Tenomodulin (Tnmd) is the mature tendon phenotype marker unique to tendon tissue. Both the PLAGA/ADSCs and PLAGA/GDF-5 groups demonstrated increased tenomodulin expression at 4 and 8 weeks (p<0.05). Ultimate tensile load strength was improved in all PLAGA groups (2, 3, and 4) versus the control. Both composite groups (2 and 3) showed improved collagen deposition, as indicated by increased Collagen Area Fraction (CAF), approaching that of normal tendon at 8 weeks (p<0.05). Scaffold resorption was evident at 4 weeks, with complete replacement of the polymer with regenerate tissue and minimal gap formation at 8 weeks without evidence of an adverse inflammatory reaction. Defects bridged using the scaffold seeded with ADSCs showed improved collagen organization and increased modulus of elasticity compared with controls as well as properties approaching those of native tendon. Discussion/Conclusions. These results demonstrate that a tubular bioresorbable scaffold can promote extracellular matrix synthesis and organization, and the formation of neo-tendinous tissue; as well as serve as a carrier of adipose stromal cells and growth factors that are effective for tendon regeneration. Cells, growth factors and synthetic biomaterial polymers may be combined as a paradigm for regenerative engineering thereby serving as promising options for improved treatments of tendon injuries and potentially improving patient outcomes

Introduction. The exact mechanisms leading to tendinopathies and tendon ruptures remain poorly understood while their occurrence is clearly associated with exercise. Overloading is thought to be a major factor contributing to the development of tendon pathologies. However, as animal studies have shown, heavy loading alone won't cause tendinopathies. It has been speculated, that malfunctioning adaptation or healing processes might be involved, triggering tendon tissue degeneration. By analysing the expression of the entirety of degrading enzymes (degradome) in pathological and non-pathological, strained and non-strained tendon tissue, the aim of this study was to identify common or opposite patterns in gene regulation. This approach may generate new targets for future studies. Materials and Methods. RNA was extracted from different tendon tissues: normal (n=7), tendinopathic (n=4) and ruptured (n=4) Achilles tendon; normal (n=4) and tendinopathic (n=4) posterior tibialis tendon; normal hamstrings tendon with or without subjection to static strain (n=4). The RNA was reverse transcribed, then pooled per group The expression of 538 protease genes was analysed using Taqman low-density array quantitative RT-PCR. To be considered relevant, changes had to be at least 4fold and measurable at a level below 36 Cts. Results. In general, there was little common regulation when exercised was compared with pathological tissue. The expression of PAMR1 and TNFαIP3 was upregulated with exercise (169-fold and 78-fold), Achilles tendinopathy (9724-fold and 7-fold) and Achilles tendon rupture (1809-fold and 10-fold), while DDI1, PSMB11 and PSH2 which were down-regulated with exercise were upregulated with Achilles pathology. Discussion. The newly found targets may deliver insights into the initiation and progression of tendon pathologies: PAMR1, a regeneration associated muscle protease which has been shown to be downregulated in Duchenne muscular dystrophy and upregulated in regenerating muscle fibers, might also be involved in tendon regeneration; TNFαIP3, which negatively regulates the NF-κB/pro-inflammatory pathway, could have anti-inflammatory function in tendon regeneration. PSMB11 and PSH2 are for the first time shown to be expressed in tendon and regulated in tendon pathology. Using this approach we were able to generate new targets and to add information on function, regulation and expression sites of recently identified proteins

Tendon and ligament tissues are fascinating in their simplistic appearance of tissue architecture coupled with outstanding biomechanical properties. In the last decade, the mechanisms governing their development, degenerative disease progression and step-wise repair process are becoming better understood. In this talk, I will present an overview of our basic research work on these following points. (i) Tendon generation: I will discuss our finding on the role of growth and biomechanical factors influencing tendon stem/progenitor cells; (ii) Tendon degeneration: I will provide evidences how disturbed cell-cell and cell-matrix contacts are involved in loss of tissue integrity; (iii) Tendon regeneration: I will present in vivo data on the application and performance of various cell populations in tendon repair

Significant challenges remain to accomplishing the development of fully functional tendon tissue substitutes that can lead to clinically effective and successful applications. Scaffolding materials must meet demanding requirements such i) mimic the hierarchical and anisotropically aligned structure of tendon tissues from the nano- up to the macroscale, ii) meet tendon mechanical requirements and non-linear biomechanical behaviour, iii) provide the necessary biophysical/biochemical cues and mechanical responsiveness to induce the tenogenic differentiation of stem cells and potentiating the effects of biochemical supplementation. On the other side, tenogenic differentiation of stem cells is still to be established, as well as the role of such cells (either naïve or pre-differentiated) in promoting tissue regeneration. We have recently found evidences that magnetic actuation can provide means of mechanically stimulating cells in a contact-free manner and, more interestingly, can also modulate inflammatory response, a critical issue for achieving tissue regeneration instead of repair. In summary, synergies of scaffold design and magnetic responsiveness can impact significantly cells behaviour as well as in vivo response and thus widen the therapeutically range of cell-laden tissue engineered constructs in tendon regeneration

Abstract. Objectives. Tendon and ligament injury poses an increasingly large burden to society. With surgical repair and grafting susceptible to high failure rates, tissue engineering provides novel avenues for treatment. This systematic review explores in vivo evidence whether mesenchymal stem cell-derived extracellular vesicles (MSC-EVs) can facilitate tendon and ligament repair in animal models. Methods. On May 26th 2021, a systematic search was performed on PubMed, Web of Science, Cochrane Library, Embase, using search terms ‘mesenchymal stem cell’ or ‘multipotent stem cell’ AND ‘extracellular vesicles’ or ‘exosomes’ AND ‘tendon’ or ‘ligament’ or ‘connective tissue’. Risk of bias was assessed using SYstematic Review Center for Laboratory animal Experimentation (SYRCLE) tool. Studies administering EVs isolated from human or animal-derived MSCs into in vivo models of tendon/ligament injury were included. In vitro, ex vivo, in silico studies were excluded, and studies without a control group were excluded. Data on isolation and characterisation of MSCs and EVs, and in vivo findings in animal models were extracted. Results. Out of 383 relevant studies, 11 case-control studies were included for data extraction, including a total of 448 animal subjects (range 10–90). Six studies utilised bone marrow-derived MSCs. All studies characterised their MSCs via flow cytometry, which expressed CD44 and CD90, and isolated EVs via ultracentrifugation (average diameter 125nm). Five studies utilised histological scoring systems, all of which reported a lower score with EV treatment, suggesting improved healing ability. Four studies reported increased anti-inflammatory cytokine expression (IL-10, TGF-β1); three studies reported decreased endogenous M1/M2 macrophage ratio with EV treatment. Eight studies reported increased maximum stiffness, breaking load, tensile strength in EV-treated tendons. Conclusion. MSC-EVs are effective therapeutic agents for tendon/ligament pathologies, attenuating the initial inflammatory response, and accelerating tendon matrix regeneration. Future randomised controlled trials are needed to definitely demonstrate MSC-EVs superiority in management of tendon/ligament injury

As we grow older, the risk of tendon degeneration and injuries increases, which can result in pain, disability, healthcare cost, and lost productivity. Even after surgical repair the results are often unsatisfactory. The cellular reasons for the differences in the healing potential, however, are not well studied. To get a deeper insight into the biological characteristics of tenocyte-like cells from different patient groups we established a biobank with material from over 150 human donors. The patients/donors suffered from rotator cuff tears and were operated to restore the function. A proportion of the isolated cells showed stem cell-like characteristics and was able to differentiate into the osteoblastic, chondrogenic and adipogenic linage. Investigating the differentiation potential of the cells with regard to donor characteristics, we were able to demonstrate that age, sex but also the “degeneration” has an impact of the cellular potential. A possibility to stimulate the cellular activity is the application of growth factors, as already clinically used for stimulation of bone healing. Therefore, the responsiveness of the cells to the growth factors Bone Morphogenetic protein-2/7 (BMP-2/7) was analysed in vitro. Independent of the donor characteristics, the cells responded to the BMP-stimulation by increased proliferation and collagen-1 synthesis. However, cells isolated from donors with high fatty infiltration of the muscle or older females were less responsive. Looking into the intracellular signalling pathway, the data showed that the BMP-signal is mainly mediated by the canonical-pathway with samd8 playing a major role. This basic research gives first information regarding the differences in tenocytes biology with respect to the donor and is important for the understanding of tendon regeneration and the future development of new treatment strategies

There is a growing socio-economic need (i.e. “ageing society”) for effective and reproducible strategies to repair musculoskeletal tissue. In particular, acute tendon injury and chronic tendinopathies remain clinically challenging and novel treatment modalities are urgently needed. Tendons resemble a connective tissue rich in highly organized collagen fibers, displaying a remarkably high tensile strength. However, partly due to the low number of cells and their more or less avascular nature tendons heal relatively slowly. Ultimately, tendon regeneration encompasses the full restoration of the biological, biochemical and biomechanical properties, which are often impaired by endogenous healing cascades. Usually, a connective scar tissue forms at the injury site and the replaced tissue does not function adequately at high strain levels, increasing the chance of re-rupture. Despite significant advancements in tissue regeneration and engineering strategies, the clinical impact for the regeneration of tendon remains limited. For the development of novel methods to repair tendons we need to pin down the molecular and cellular mechanisms amenable to modulate endogenous (or exogenous) cell behaviour towards functional tissue regeneration. By comparing the gene expression profile of Achilles tendon tissue harvested from young-mature and old mice we demonstrate profound changes in the expression of ECM-related proteins and a previously unknown role of Secreted protein acidic and rich in cysteine (Sparc; also known as BM-40 or osteonectin) in tendons. Sparc levels in tendons are critical for proper collagen fibril maturation and its age-related decrease, together with a change in ECM properties potentially drives adipogenic differentiation of tendon stem and progenitor cells (TDSPCs) and consequently lipid accretion in tendons. Generally, the fate of stem/ progenitor cells is largely determined by stimuli from the stem cell niche. In tendons, we describe a novel cellular barrier, most likely preventing the leakage of blood-borne products into the tendon proper. We propose that this “blood-tendon barrier” is part of the stem cell niche in tendons controlling TDSCP fate, preventing erroneous differentiation. By investigating the developmental programs driving tendon tissue formation and on the other hand the mechanisms contributing to the senescence of tendons, ultimately resulting in decreased quality of tendons in the elderly, novel targets for clinical intervention potentially can be discovered

Metabolic disorders are frequently associated with tendon degeneration and impaired healing after acute injury. However, the underlying cellular and molecular mechanisms remain largely unclear. We have previously shown that human and rat tendon cells responde to glucose stimulation in vitro by secretion of insulin. Therefore, we now hypothesize that nutritional glucose uptake affects tendon healing in a rat model. In female rats (n=30/group), unilateral full-thickness Achilles tendon defects were created. Immediately after surgery animals were either fed a glucose rich- or a control diet for up to 4 weeks. Gait analysis (Catwalk, Noldus) was performed at three time points. In addition, tendon thickness measurements, biomechanical testing and immunohistochemical analysis were conducted. Subsequently, gene expression analysis, comparing cDNA pools (n=5) prepared from repair tissues of both groups was performed. The repair tissues of the high glucose group were significantly thicker compared to the control group (p<0.001). The intermediate toe spread, an indicator of pain, were significantly improved in the high glucose group one and two weeks post surgery. Biomechanical analysis revealed that the repair tissues of the high glucose group were significantly stiffer (p<0.05) compared to the control group, no significant difference was detected for maximum tensile load…. The proportion of Ki67+ cells in the repair tissue was 3.3% in the control diet group and 9,8% in the high glucose group, indicating increased cell proliferation (p<0.001). Finally, gene expression analysis revealed the chondrogenic marker genes Collagen II, Aggrecan, COMP and SOX9 to be upregulated and genes involved in lipid metabolism like PPARgamma and Fabp2 to be downregulated in the glucose diet group. Here we show fort he first time that a high-glucose diet affects gait pattern and tendon biomechanics, influences tendon thickness and cell proliferation. Gene expression analysis reveals a regulation of chondrogenic as well as adipogenic marker genes. The molecular mechanisms underlying these effects on cells and extracellular matrix are currently under investigation, potentially revealing targets for developing a dietary intervention scheme to support tendon regeneration after trauma or tendon disease

Introduction. Tendon cells originate from yet poorly described precursor cells and develop in a particular “niche” close to vascular walls. Several factors have been described to determine this niche such as mechanical stimuli, oxygen tension, composition and structure of the extracellular matrix (ECM). Also, the vasculature is considered to play a crucial role for tendon cell development, yet evidence of how this is accomplished is lacking. In this study we therefore focussed on the endothelium of tendon vessels postulating the existence of a paracellular barrier. Materials and Methods. By electron microscopy, immunohistochemistry, and RT-PCR we investigated the presence of constituents making up such an endothelial barrier which we subsequently tested for its functionality by tracer injection. Moreover, we performed differentiation experiments into the adipogenic, chondrogenic and osteogenic lineage on tendon derived cells in the presence and absence of serum. Expression levels and activity of matrixmetalloproteinases (MMPs) were assessed by western blot and zymography. Results. Perfusion with defined tracer substances revealed that the blood-tendon-barrier impedes the passive transport of macromolecules (>10 kDa) from the blood stream to the surrounding tendon tissue, but is permeable to molecules <287D. The expression of barrier-related proteins, such as zonula occludens protein-1 (ZO-1), occludin, claudin-3, and claudin-5, in human and murine tendon vascular cells further corroborates the assumption that a restrictive tissue barrier acts at the blood-tendon interface. In vitro experiments indicate that serum enhances the number of adipocytes and increases the amount of calcium deposits as demonstrated by Oil Red O and Alizarin S staining, respectively. Also, serum appears to significantly raise the expression level of MMP2 and MMP9 known to remodel the ECM. Discussion. Here, we postulate the existence of a blood-tendon barrier, which separates tendon tissue from the systemic circulation in intact tendons and, thus, impedes the paracellular passage of blood-borne molecules >10kD. In vitro studies investigating the influence of serum on tendon derived cells show that serum considerably modifies the differentiation potential and increases the expression of matrix remodelling enzymes. The role of this structure in tendinopathy, tendon regeneration and tendon development remains to be elucidated

Tendon regeneration is complex since the scaffold has to bear high loads and stress concentrations, while providing suitable deformability. Previous studies demonstrated a physiological orientation of the fibers and good cell adhesion on electrospun polymeric scaffolds [1]. The aims of this work were to: (i) prepare and characterize electrospun resorbable scaffolds with different compositions and (ii) develop a process to produce a multiscale bundle assembly to mimic the hierarchical structure and biomechanical properties of a real tendon. We produced fibrous scaffolds made of blends of poly-L-lactic acid (PLLA) and collagen (Coll):. Pure PLLA;. PLLA/Coll 75/25 w/w;. PLLA/Coll 50/50 w/w. In order to prepare 3D bundles made of aligned fibres, we used a high-speed rotating collector. The electrospun nanofibers were deposited tangentially onto the drum, the electrospun layer was manually rolled transversely along the drum and then removed. The bundles were approximately 150 mm long and 300–450 mm in diameter. Five specimens were prepared and tested for each blend. To evaluate the mechanical properties of the bundles a tension test was applied with capstan grips on a testing machine with a 100N load cell, under the following conditions:. Gauge length: 20 mm. Monotonic ramp to break detection. Actuator speed 5 mm/min. For all the bundles, the stress-strain curve showed an initial non-linear part (toe region), similar to the laxity of the tendon at rest. The mechanical analysis confirmed the outstanding ductility and toughness of pure PLLA. Increasing the percentage of collagen resulted in a reduction of ductility. The PLLA/Coll 50/50 had a rather brittle behaviour. The values of mechanical properties found for the different compositions were slightly lower but of the same order of magnitude as tendon fibers (Failure stress: 33.7±19.2 MPa; Failure strain: 21.0±9.1 %; Young Modulus: 257±101 MPa [2]). The bundles made of pure PLLA had a failure stress of 13.2±0.8 MPa; failure strain of 84.7±9.4%; Young Modulus of 78.6±7.5 MPa. The bundles made of PLLA/Coll 50/50 had: failure stress of 10.5±1.5 MPa; failure strain of 21.4±2.7%, Young Modulus of 65.7±9.8 MPa. The most promising composition was the PLLA/Coll 75/25, with a failure stress of 14.0±0.7 MPa; failure strain of 40.3±2.2 %, Young Modulus of 98.6±12 MPa. We also tested bundles mechanical properties after aging samples in phosphate buffer at 37 °C for 48 hours, 7 and 14 days. After ageing, stress and strain values were progressively lower, while the toughness increased, compared to the dry samples. The promising results found in this work for the electrospun PLLA-collagen blends confirm their potential use for tendon tissue regeneration. This is a starting point for developing multiscale scaffolds mimicking the structure of tendon tissue, which can potentially be used in human regenerative medicine both as bioresorbable prosthesis, or inserted in a bioreactor for in vitro production of tendon tissue