Objectives. Re-rupture is common after primary flexor tendon repair. Characterization of the biological changes in the ruptured tendon stumps would be helpful, not only to understand the biological responses to the failed tendon repair, but also to investigate if the tendon stumps could be used as a recycling biomaterial for
Aims. To evaluate the donor site morbidity and tendon morphology after harvesting whole length, full-thickness peroneus longus tendon (PLT) proximal to the lateral malleolus for ligament reconstructions or tendon transfer. Methods. A total of 21 eligible patients (mean age 34.0 years (standard deviation (SD) 11.2); mean follow-up period 31.8 months (SD 7.7), and 12 healthy controls (mean age, 26.8 years (SD 5.9) were included. For patients, clinical evaluation of the donor ankle was performed preoperatively and postoperatively. Square hop test, ankle strength assessment, and MRI of distal calf were assessed bilaterally in the final follow-up. The morphological symmetry of peroneal tendons bilaterally was evaluated by MRI in healthy controls. Results. Among the patients, the mean pre- and postoperative American Orthopaedic Foot and Ankle Society (AOFAS) hindfoot score and Karlsson-Peterson score were 98.7 (SD 2.5; p = 0.480) and 98.5 (SD 2.4; p = 0.480), and 98.3 (SD 2.4; p = 0.162) and 97.9 (SD 2.5; p = 0.162), respectively. There was no significant difference between square hop test bilaterally (p = 0.109) and plantar flexion peak force bilaterally (p = 0.371). The harvested limb had significantly less eversion peak force compared to the contralateral limb (p < 0.001). Evidence of probable
Objectives. The objective of this study was to determine if the use of fascia lata as a
Anterior cruciate ligament reconstruction (ACLR) using a semitendinosus (ST) autograft, with or without gracilis (GR), results in donor muscle atrophy and varied
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
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 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
Introduction. Supraspinatus tears comprise most rotator cuff injuries, the leading cause of shoulder pain and an increasing problem with ageing populations. Surgical repair of considerable or persistent damages is customary, although not invariably successful. Tissue engineering presents a promising alternative to generate functional tissue constructs with improved healing capacities. This study explores tendon tissue constructs’ culture in a platform providing physiological mechanical stimulation and reports on the effect of different loading regimes on the viability of human tendon cells. Method. Porcine decellularized tendon scaffolds were fixed into flexible, self-contained bioreactor chambers, seeded with human tenocytes, allocated in triplicates to either static control, low (15±0.8Newtons [N]), medium (26±0.5N), or high (49±2.1N)-force-regime groups, connected to a perfusion system and cultured under standard conditions. A humanoid robotic arm provided 30-minute adduction/abduction stimulation to chambers daily over a week. A metabolic activity assay served to assess cell viability at four time points. Statistical significance = p<0.05. Result. One day after beginning mechanical stimulation, chambers in the medium and high-force regimes displayed a rise in metabolic activity by 3% and 5%, respectively. By the last experimental day, all mechanical stimulation regimes had induced an augment in cell viability (15%, 57% and 39% with low, medium, and high loads, respectively) matched against the static controls. Compared to all other conditions, the medium-force regime prompted an increased relative change in metabolic activity for every time point set against day one (p<0.05). Conclusion. Human tenocytes’ viability reflected by metabolic activity in a physiologically relevant bioreactor system is enhanced by loading forces around 25N when mechanically stimulating using adduction/abduction motions. Knowing the most favourable load regime to stimulate tenocyte growth has informed the ongoing exploration of the distinctive effect of different motions on
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
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
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 injuries are a worldwide problem affecting several age groups and stem cell based therapies hold potential for tendon strategies guiding
Introduction. The rabbit common calcanean (Achilles) tendon is a compound apparatus frequently used in studies considering novel interventions to facilitate
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
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
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 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)
Cellular therapies play an important role in tendon tissue engineering with tenocytes being described as the most prominent cell population if available in large numbers. However, in vitro expansion of tenocytes in standard culture leads to phenotypic drift and cellular senescence. Recent work suggests that maintenance of tenogenic phenotype in vitro can be achieved by recapitulating different aspects of the native tendon microenvironment. One approach used to modulate the in vitro microenvironment and enhance extracellular matrix (ECM) deposition is macromolecular crowding (MMC). MMC is based on the addition of inert macromolecules to the culture media mimicking the dense extracellular matrix. In addition, as tendon has been described to be a relatively avascular and hypoxic tissue and low oxygen tension can stimulate collagen synthesis and cross-linking, we venture to assess the synergistic effect of MMC and low oxygen tension on human tenocyte phenotype maintenance by enhancing synthesis and deposition of tissue-specific ECM. Human tendons were kindly provided from University Hospital Galway, after obtaining appropriate licenses, ethical approvals and patient consent. Afterwards, tenocytes were extracted using the migration method. Experiments were conducted at passage three. Optimization of MMC conditions was assessed using 50 to 500 μg/ml carrageenan (Sigma Aldrich, UK). For variable oxygen tension cultures, tenocytes were incubated in a Coy Lab (USA) hypoxia chamber. ECM synthesis and deposition were assessed using SDS-PAGE (BioRad, UK) and immunocytochemistry (ABCAM, UK) analysis. Protein analysis for Scleraxis (ABCAM, UK) was performed using western blot. Gene analysis was conducted using a gene array (Roche, Ireland). Cell morphology was assessed using bright-field microscopy. All experiments were performed at least in triplicate. MINITAB (version 16, Minitab, Inc.) was used for statistical analysis. Two-sample t-test for pairwise comparisons and ANOVA for multiple comparisons were conducted. SDS-PAGE and immunocytochemistry analysis demonstrated that human tenocytes treated with the optimal MMC concentration at 2% oxygen tension showed increased synthesis and deposition of collagen type I, the major component of tendon ECM. Moreover, immunocytochemistry for the tendon-specific ECM proteins collagen type III, V, VI and fibronectin illustrated enhanced deposition when cells were treated with MMC at 2% oxygen tension. In addition, protein analysis revealed elevated dexpression of the tendon-specific protein Sclearaxis, while a detailed gene analysis revealed upregulation of tendon-related genes and downregulation of trans-differentiation markers again when cells cultured with MMC at 2% oxygen tension. Finally, low oxygen tension and MMC did not affect the metabolic activity, proliferation and viability of human tenocytes. Collectively, results suggest that the synergistic effect of MMC and low oxygen tension can accelerate the formation of ECM-rich substitutes, which stimulates tenogenic phenotype maintenance. Currently, the addition of substrate aligned topography together with MMC and hypoxia is being investigated in this multifactorial study for the development of an implantable device for
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
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
