Worldwide, tendon disorders are one of the main causes of disability that decrease the quality of life of individuals and represent a substantial economic burden on society. Currently, the main therapies used for tendon injuries are not able to restore tendon functionality, and due to tendons' hypovascular and hypocellular nature, they present a reduced healing capacity, which also limits the success of the available therapies. In order to discover new therapies, extracellular vesicles (EVs), key players in cell-cell communication, have been widely explored for tissue engineering and regenerative medicine applications. Thus, the aim of this study is to assess the role of EVs derived from platelets in stem cell tenogenic commitment using a bioengineered tendon in vitro model for potential use as tendon therapeutic agents. Biomimetic platelet-derived EVs were produced by freeze-thaw cycles of platelets and isolation at different centrifugation speed. To recreate the architecture of tendons, a 3D system consisting of electrospun anisotropic nanofiber scaffolds coated with collagen encapsulating human adipose stem cells (hASCs) and different types of platelet-derived EVs, were produced. Then, the influence of the tendon-mimetic constructs and the distinct EVs populations in the hASCs tenogenic differentiation were assessed over culture time. We observed that the hASCs on the nanofibrous tendon scaffolds, show high cytoskeleton anisotropic organization that is characteristic of tenocytes. Moreover, acting as biological cues, platelet-derived EVs boosted hASCs tenogenic commitment, supported by the increased gene expression of tendon-related markers (SCX and TNMD). Additionally, EVs enhanced the deposition of tendon like extracellular matrix (ECM), as evidenced by the increased gene expression of ECM-related markers such as COL1, COL3, DCN, TNC, and MMP-3, which are fundamental for ECM synthesis and degradation balance. Moreover, EVs induced lower collagen matrix contraction on hASCs, which has been related with lower myofibroblast differentiation. Overall, the results revealed that EVs are capable of modulating stem cells' behavior boosting their tenogenic commitment, through the increased expression of healthy tendon cell markers, potentiating ECM deposition and decreasing cell contractility. Therefore, platelet EVs are a promising biochemical tool, worthy to be further explored, as paracrine signaling that might potentiate tendon repair and regeneration.

Meniscus tears have been treated using partial meniscectomy to relieve pain in patients, although this leads to the onset of early osteoarthritis (OA). Cell-based therapies can help preserve the meniscus, although the presence of inflammatory cytokines compromises clinical outcomes. Anti-inflammatory drugs (e.g. celecoxib), can help to reduce pain in patients and in vitro studies suggest a beneficial effect on cytokine inhibited matrix content. Previously, we have demonstrated that the inhibitory effects of IL-1β can be countered by culture under low oxygen tension or physioxia. The present study sought to understand whether physioxia, celecoxib or combined application can counter the inhibitory effects IL-1β inhibited meniscus cells.

Human avascular and vascular meniscus cells (n =3) were isolated and expanded under 20% (hyperoxia) or 2% (physioxia) oxygen. Cells were seeded into collagen scaffolds (Geistlich, Wolhusen) and cultured for 28 days either in the presence of 0.1ng/mL IL-1β, 5µg/mL celecoxib or both under their expansion oxygen conditions. Histological (DMMB, collagen I and collagen II immunostaining), GAG content and gene expression analysis was evaluated for the scaffolds.

Under hyperoxia, meniscus cells showed a significant reduction in GAG content in the presence of IL-1β (*p < 0.05). Celecoxib alone did not significantly increase GAG content in IL-1β treated cultures. In contrast, physioxic culture showed a donor dependent increase in GAG content in control, IL-1β and celecoxib treated cultures with corresponding histological staining correlating with these results. Additionally, gene expression showed an upregulation in COL1A1, COL2A1 and ACAN and a downregulation in MMP13 and ADAMTS5 under physioxia for all experimental groups.

Physioxia alone had a stronger effect in countering the inhibitory effects of IL-1β treated meniscus cells than celecoxib under hyperoxia. Preconditioning meniscus cells under physioxia prior to implantation has the potential to improve clinical outcomes for cell-based therapies of the meniscus.

Partial meniscectomy patients have a greater likelihood for the development of early osteoarthritis (OA). To prevent the onset of early OA, patient-specific treatment algorithms need to be created that predict patient risk to early OA after meniscectomy. The aim of this work was to identify patient-specific risk factors in partial meniscectomy patients that could potentially lead to early OA.

Partial meniscectomy patients operated between 01/2017 and 12/2019 were evaluated in the study (n=317). Exclusion criteria were other pathologies or surgeries for the evaluated knee and meniscus (n = 114). Following informed consent, an online questionnaire containing demographics and the “Knee Injury and Osteoarthritis Outcome Score” (KOOS) questionnaire was sent to the patient. Based on the KOOS pain score, patients were classified into “low” (> 75) and “high” (< 75) risk patients, indicating risk to symptomatic OA. The “high risk” patients also underwent a follow-up including an MRI scan to understand whether they have developed early OA.

From 203 participants, 96 patients responded to the questionnaire (116 did not respond) with 61 patients considered “low-risk” and 35 “high-risk” patients. Groups that showed a significant increased risk for OA were patients aged > 40 years, females, overweight (BMI >25 kg/m2 ≤ 30 kg/m2), and smokers (*p < 0.05). The “high-risk”-follow-up revealed a progression of early osteoarthritic cartilage changes in seven patients, with the remaining nineteen patients showing no changes in cartilage status or pain since time of operation. Additionally, eighteen patients in the high-risk group showed a varus or valgus axis deviation.

Patient-specific factors for worse postoperative outcomes after partial meniscectomy and indicators for an “early OA” development were identified, providing the basis for a patient-specific treatment approach. Further analysis in a multicentre study and computational analysis of MRI scans is ongoing to develop a patient-specific treatment algorithm for meniscectomy patients.

The goal was to analyze the cellular response, specifically the osteogenic capacity, of titanium (Ti) implants harbouring a novel laserbased-surface-structure with the overall aim: augmented osteointegration. Surface micro-/nanoproperties greatly influence cell behaviour at the tissue-implant-interface and subsequent osteointegration. We investigated Ti-materials subjected to a specially developed shifted-Laser-Surface-Texturing (sLST) technology and compared them to a standard roughening-technique (sand-blasting-acid-etching, SLA). The biological response was evaluated with hMSCs, which are naturally available at the bone-implant-interface. We hypothesized: the novel surface is beneficial for our three different (young/healthy-YH; aged/healthy-AH;aged/osteoporotic-OP) cohorts.

The sLST was performed using a SPI-G3-series laser (beam-wavelength=1064nm, pulse-duration=200ns). For the SLA surface, Ti was sandblasted, afterwards acid-etched (HCl/H2SO4). Three different hMSC cohorts were studied: YH: n=6,29±6; AH: n=5,79±5; OP: n=5,76±5 years (osteoporosis confirmed via DEXA-scan). OP hMSCs show e.g. ColI-deficient-matrix and decreased mineralization. Cells were examined for survival, cell proliferation and cytoskeleton arrangement. Osteogenic differentiation was carried out over 21 days, matrix mineralization was validated with Alizarin-Red-S-staining and quantification.

Laser-texturing generated precisely the desired microgeometry. On nanostructural level, differently-sized Ti-droplets were formed stochastically by laser-induced-Ti-plasma. Live/dead-/Actin-stainings showed comparable results for all cohorts and surfaces in terms of survival and cell shape. On Ti-materials, cell growth showed no significant difference between the 3 cohorts. Alizarin quantification revealed the highest levels on laser-textured-surfaces; highest value for YH, followed by AH, lastly OP; no significance between AH/YH, but between OP/YH (p<0.0001). However, mineralization of all cohorts cultured on laser-textured-surfaces increased significantly (p<0.0001) compared to respective SLA-group, with >20fold higher value in the OP-cohort (AH:11fold, YH:6fold).

The data proves the biocompatibility of the laser-structured-Ti for young+aged cohorts. Osteogenic differentiation was significantly augmented on laser-treated-Ti. Most intriguingly, OP-donors could reach manifold increased mineralization, suggesting the novel laser texturing can counteract the osteoporotic phenotype. As osteogenesis-enhancing capacities may be related to mechanisms controlling cellular shape/fate, further investigations referring to this are currently ongoing. In conclusion, our laser-textured-Ti-materials are safe, can have a demand-oriented designer-surface-topography and represent a great potential for development into next-generation-implants suitable for different patient-cohorts, especially osteoporosis patients.

Introduction and Objective

The meniscus is composed of two distinct regions, a vascular outer zone and an avascular inner zone. Due to vascularization, tears within the vascular zone can be treated by suturing. However, tears in the avascular zone have a poor healing capacity and partial meniscectomy is used to prevent further pain, although this leads to early osteoarthritis. Previous studies have demonstrated that the vascular zone contains a progenitor population with multilineage differentiation potential. Isolation and propagation of these progenitors can be used to develop cell-based therapies for treating meniscal defects. In vivo, the meniscus resides under a low oxygen environment, also known as physioxia (2–7% oxygen) and previous work suggests that it promotes the meniscal phenotype. The objective of the study was to isolate progenitor populations from both meniscus regions and to examine their clonogenecity and differentiation potential under both hyperoxia (20% oxygen) and physioxia (2% oxygen). We hypothesize that physioxia will have a beneficial effect on colony formation and trilineage differentiation of meniscal cells.

Osteoarthritis (OA) is a progressive and degenerative joint disease resulting in changes to articular cartilage. In focal early OA defects, autologous chondrocyte implantation (ACI) has a 2-fold failure rate due to poor graft integration and presence of inflammatory factors (e.g. Interleukin-1β). Bone marrow derived mesenchymal stem cells (MSCs) are an alternative cell source for cell-based treatments due to their chondrogenic capacity, though in vivo implantation leads to bone formation. In vivo, chondrocytes reside under an oxygen tension between 2–7% oxygen or physioxia. Physioxia enhances MSC chondrogenesis with reduced hypertrophic marker (collagen X and MMP13) expression compared to hyperoxic conditions (20% oxygen). This study sought to understand whether implantation of physioxic preconditioned MSCs improves cartilage regeneration in an early OA defect model compared to hyperoxic MSCs. Bone marrow extracted from New Zealand white rabbits (male: 5–6 months old; n = 6) was split equally for expansion under 2% (physioxia) or 20% (hyperoxia) oxygen. Chondrogenic pellets (2 × 105 cells/pellet) formed at passage 1 were cultured in the presence of TGF-β1 under their expansion conditions and measured for their wet weight and GAG content after 21 days. During bone marrow extraction, a dental drill (2.5mm diameter) was applied to medial femoral condyle on both the right and left knee and left untreated for 6 weeks. Following this period, physioxia and hyperoxia preconditioned MSCs were seeded into a hyaluronic acid (TETEC) hydrogel. Fibrous tissue was scraped and then MSC-hydrogel was injected into the right (hyperoxic MSCs) and left (physioxia MSCs) knee. Additional control rabbits with drilled defects had fibrous tissue scrapped and then left untreated without MSC-hydrogel treatment for the duration of the experiment. Rabbits were sacrificed at 6 (n = 3) and 12 (n = 3) weeks post-treatment, condyles harvested, decalcified in 10% EDTA and sectioned using a cryostat. Region of interest was identified; sections stained with Safranin-O/Fast green and evaluated for cartilage regeneration using the Sellers scoring system by three blinded observers. Physioxic culture of rabbit MSCs showed significantly shorter doubling time and greater cell numbers compared to hyperoxic culture (∗p < 0.05). Furthermore, physioxia enhanced MSC chondrogenesis via significant increases in pellet wet weight and GAG content (∗p < 0.05). Implantation of physioxic preconditioned MSCs showed significantly improved cartilage regeneration (Mean Sellers score = 7 ± 3; ∗p < 0.05) compared to hyperoxic MSCs (Sellers score = 12 ± 2) and empty defects (Sellers score = 17 ± 3). Physioxia enhances in vitro rabbit MSC chondrogenesis. Subsequent in vivo implantation of physioxia preconditioned MSCs improved cartilage regeneration in an early OA defect model compared to hyperoxic MSCs. Future studies will investigate the mechanisms for enhanced in vivo regeneration using physioxia preconditioned MSCs.

Numerous implanted hip and knee joint arthroplasties have to be replaced due to early or late loosening of the implant, a failure of osteointegration with fibrous tissue at the bone-implant-interface. This could be counteracted by ensuring that cells which attach to the implant surface differentiate towards bone cells afterwards. For this reason, human mesenchymal stem cells (hMSCs) will be included in this study. These cells are naturally available at the bone-implant-interface, multipotent and therefore ideal to study the osteoinductivity of a material. The goal of this pilot study was to test the cell response towards three different titanium grades with a novel surface structuring, as a first step towards achieving an improved implant surface for enhanced osteointegration. Disk-shaped titanium scaffolds with a diameter of 12 mm and a height of 1.2 mm were used. The surface topography (500 µm × 500 µm × 300 µm pores) was generated via laser treatment of the surface. By using nanosecond pulsed laser technique, a rough surface with micro- and nanostructural (titanium droplets) features was automatically formed. Three different batches made of commercially pure titanium grades 1 and 2 (Ti1/Ti2) or Ti6Al4V alloy grade 5 (Ti5) were produced. Four cell types were analysed on these batches: primary hMSCs from one donor (m, 25 y), periosteum derived cells (PDCs), human osteoblasts (hOBs) and periodontal ligament cells (PDLs). Cells were seeded on Ti1, Ti2 and Ti5 scaffolds in triplicates. Resazurin assay to examine cell viability was conducted with all cell types. Measurements were executed on several days after seeding, from day one up to day 14. Actin staining as well as live/dead staining was performed with hMSCs cultured on titanium for 1, 3, 5 or 7 days. The cell viability assay revealed early turning points of growth for osteogenic hOBs (day 3) and PDCs (day 7). HMSCs grew steadily on the material and non-osteogenic PDLs stayed in plateau throughout the cultivation period. With respect to the material, cells demonstrated better proliferation on Ti1 and Ti2 than on Ti5. Live/dead staining showed a high survival rate of hMSCs at each time point and on all three titanium grades, with a neglectable number of dead cells. Actin staining confirmed an enhanced spreading and stretching of hMSCs on Ti1 and Ti2 compared to hMSCs on Ti5. Our pilot data indicates that cells react to different titanium compositions, revealed by increased proliferation on commercially pure titanium (Ti1/2). Furthermore, our results demonstrate that osteogenic cells prefer the novel surface structuring in comparison to non-osteogenic PDL cells, which stayed in plateau. The turning points of growth (hOBs/PDCs) suggest an osteosupportiveness of the surface. Although hMSCs did not show a turning point in growth, their growth was steady and resulted in the highest number of cells along with a well stretched morphology. Due to their good proliferation and response to the material, hMSCs are currently being used for evaluating the osteogenic potential of the novel scaffolds.

Introduction

Cell-based tendon engineering is an attractive alternative therapeutic approach to established treatments of tendon injuries. Numerous cell types are promising source of tendon engineering; however, there are certain disadvantages for each cell type. Interestingly, dermal fibroblasts (DFs) are able to transdifferentiate into other cell types, they are widely distributed in dermis and easy to harvest and isolate. Furthermore, pilot clinical studies suggested a promising therapeutic potential of autologous DFs for discorded tendons (Connell et al., 2009&2011), but the underlining repair mechanisms remain unclarified. To investigate tenogenic differentiation process in great detail, we have previously established a three-dimensional (3D) cell sheet model, comprising of three consecutive step (expansion, stimulation and maturation) leading to the formation of 3D tendon-like tube (Hsieh et al., 2018; Yan et al., 2020). Hence, the aim of this study was to carry out pilot examination of the tenogenic potential of human DFs (hDFs) by implementing the 3D cell sheet model.

Background: The exact pathways of collagen remodeling in tendon tissue are not well understood. Therefore, we have established a 3D collagen gel system and studied the remodeling capacity of two different TSPC lines: young, Y-TSPC and aged/degenerative, A-TSPC. We specifically investigated the involvement of integrin receptors in the remodeling process. Methods: Y- and A-TSPC were derived from human Achilles tendon. RT-PCR was used to assess the expression of collagen-binding integrins. Integrins a1 and a11 were silenced by lentiviral delivery of shRNA in the Y-TSPC. Control-shRNA, a1-shRNA and a11-shRNA virus was given for 24h and then cells were selected with zeocin for 10 days. The integrin knockdown (KD) efficiency was assessed by quantitative PCR and western blotting. Last, time-lapse recording of gel contraction of Y-TSPC+con, Y-TSPC+a1KD, Y-TSPC+a11KD, and A-TSPC were performed. Results: Integrin a1 and a11 were significantly downregulated in A-TSPC. Therefore, to mimic the A-TSPC we carried out a1 and a11 KD in Y-TSPC. PCR and western blot validated very efficient KD. Analyses of collagen contraction revealed that Y-TSPC+a11KD had significant reduction in collagen contractibility comparable to A-TSPC phenotype. Regarding integrin a1, we found that this receptor had no effect on the contraction rate of TSPC. Thus, to our knowledge we have now identified for the first time a novel role of a11 integrin in tendon matrix remodeling, and a follow up analyses of the exact downstream cascade are on the way.

The establishment of a proper musculoskeletal system depends on the well-organized and synchronized development of muscle, tendon and cartilage/bone. In tendon biology, a great progress in identifying tendon-specific genes (Scleraxis, Mohawk, Tenomodulin) had been made in the last decade. However, there are many open questions regarding the exact function of genes in tendon development and homeostasis. The purpose of this study was to perform a systematic review of publications describing tendon-related genes, which were studied in-depth and characterized by using knockout technologies and the respectively generated transgenic mouse. Method: Literature search was carried out in Pubmed using “tendon” and “mouse knockout” and “phenotype” and was not limited to year. Results: We report in a tabular manner, that from a total of 25 tendon-related genes, in 23 of the respective knockout mouse models phenotypic changes were detected. Additionally, in some of the models it was described at which developmental stages these changes appeared and progressed. Interestingly, so far only loss of Scleraxis and TGFbeta signaling led to severe tendon developmental phenotypes, while mice deficient for various proteoglycans, Mohawk, EGR1 and 2, and Tenomodulin exhibited mild phenotypes. This suggests that in general the tendon developmental program is well backup and specifically that among the members of the proteoglycan family there are clear compensatory effects. In future, it will be of great importance to discover additional master tendon transcription factors as well as genes that play indispensable roles in tendon development.

Tendons are dense connective tissues and critical components of the musculoskeletal system with known long repair process. Tissue engineering is a promising approach for achieving complete recovery of ruptured tendons. The most studies have focused on the combination of cells with various carriers; however, frequent times the biomaterials do not match the tissue organization and strength. For this reason, we first reviewed the literature for an alternative scaffold-free strategy for tendon engineering and second, we compared the cell sheet formation of two different cell types: bone marrow-derived mesenchymal stem cells (BM-MSCs) and tendon stem/progenitor cells (TSPCs). Methods: Literature search was performed in Pubmed using “tendon tissue engineering” and “scaffold-free” keywords and was limited to the last ten years. By using a 3-step protocol, BM-MSCs and TSPCs were induced to form cell sheets in 5 weeks. The sheets were compared by analysis for weight, diameter, cell density, tissue morphology (H&E and scoring) and cartilaginous matrix (DMMB and S.O. staining). Results: Scaffold-free models (cell sheets and pellet cultures) are available; however, further optimization is needed. Comparison between the two cell types clearly demonstrated that TSPCs form more mature cell sheet, while BMSCs form larger but less organized and differentiated sheet as judged by higher cell density and lower scoring outcome. Future efforts will focus on identifying mechanisms to speed BM-MSC sheet formation and maturation, which can in turn lead to development of new methodology for scaffold-free tendon tissue engineering.

Mesenchymal Stem Cells (MSCs) are a candidate cell type for treating osteoarthritic focal defects. In vivo, cartilage and bone marrow reside under a low oxygen tension, between 2–7% oxygen or physioxia, that has been shown to enhance MSC chondrogenesis. However, chondrogenesis is inhibited in the presence of IL-1. Here, it was hypothesized that physioxia reduces IL-1 inhibited chondrogenesis. Human MSCs (Mean age, 32 years; n = 9) were split equally for expansion under either 2% (physioxia) or 20% (hyperoxia) oxygen. Chondrogenic pellets (2 × 10⁵ MSCs/pellet) were formed and cultured in the presence of 10 ng/ml TGF-b₁ and in combination with either 0.1 or 0.5 ng/ml IL-1 under their respective expansion conditions. Pellets were assessed for their wet weight, GAG and collagen II content and evaluated histologically (Collagen X and MMP-13). Statistical analysis was performed using a Two-way ANOVA with Tukey post-hoc test, significant differences stated when p < 0.05. A significant dose-dependent IL-1 inhibition in chondrogenesis was observed for pellet wet weight and GAG content under hyperoxia (p < 0.05). Physioxia alone significantly increased wet weight, GAG and collagen II content (p < 0.05) compared to hyperoxia. A donor-dependant response was observed, whereby 80% of donors responded to physioxia and their analysis showed significant increases in wet weight and GAG content in the presence IL-1(p < 0.05). Furthermore, reduced hypertrophy marker expression (Collagen X and MMP-13) was observed under physioxia in the presence of IL-1. The molecular signalling mechanisms controlling these responses are to be investigated.

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.

Introduction

Human Mesenchymal stem cells (hMSCs) are a promising source for articular cartilage repair. Unfortunately, under in vitro conditions, chondrogenically differentiated hMSCs have the tendency to undergo hypertrophy similar to growth plate chondrocytes. Retinoic acid (RA) signalling plays a key role in growth plate hypertrophy. Whilst RA agonists block chondrogenesis and foster hypertrophy during later stages, RAR inverse agonists (IA) enhance chondrogenesis when applied early in culture. Therefore, we hypothesized that treatment with RAR IA will attenuate hypertrophy in chondrogenically differentiated hMSCs. To test this hypothesis, we analysed early (initial chondrogenic differentiation) and late treatment (hypertrophy stage) of hMSCs with an RAR IA.

Osteoarthritis is a degenerative disease mainly caused by aging, although in younger patients (aged 25 – 50) it can be a consequence of sports-related injuries or trauma. This results in early osteoarthritis with subsequent changes in cartilage extracellular matrix. Cell-based tissue engineering approaches using mesenchymal stem cells (MSCs) are an ideal cell type for the treatment of early osteoarthritc defects. Our group has demonstrated in a clinical study, that interleukin-1β (IL-1β) was expressed in cartilage plugs from patients with early osteoarthritis. In vitro studies have shown that IL-1β inhibits cartilage formation in chondrocytes or MSCs undergoing chondrogenesis. However, these studies show complete inhibition of tissue formation, whereas in the context of early osteoarthritis, cartilage extracellular matrix remains around the defect site. Thus, the present study sought to develop a model mimicking early osteoarthritis using MSCs.

Osteoarthritis is a degenerative disease that results in changes in cartilage extracellular matrix. In vitro studies have shown that IL-1β inhibits cartilage formation in chondrocytes or MSCs undergoing chondrogenesis. In vivo, articular chondrocytes and bone marrow reside under hypoxic or physioxic environment (1–5% oxygen) and previous investigations have shown an increase in cartilage matrix proteins and reduced hypertrophy for MSC chondrogenesis, especially for MSCs expanded and differentiated under physioxia. Our hypothesis was that physioxic preconditioning reduces the effects of IL-1β inhibited MSC chondrogenesis.

Introduction

Modulation of signaling pathways, which involves tendon development, regeneration, or homeostasis, is one of the potential modalities to facilitate proper regeneration of the injured tendon. Authors have previously reported that activation of Wnt/beta-catenin signaling suppressed the expression of tenogenic genes (i.e. Scleraxis (Scx), Mohawk (Mkx), Tenomodulin (Tnmd)) in rat primary tendon-derived cells (TDCs) and SCX-transduced human mesenchymal stem cells (hMSC-Scx cells), as a tendon progenitor cell line (kindly provided Dr. Docheva). The roles of TGF-beta signaling in tenogenesis have been elucidated. The purpose of the study was to evaluate the effect of TGF-beta signaling on tenogenic genes and relationship between both two signalings in rat TDCs and hMSC-Scx cells.

The exact pathways of collagen remodeling in tendon tissue are not well understood. Therefore, we have established an ex vivo 3D collagen gel-based system and we studied the remodeling capacity of two different TSPC lines from young, Y-TSPC and aged/degenerative, A-TSPC donors. Here, we specifically focused on investigating the involvement of integrin receptors in the remodeling process. Integrins are transmembrane receptors consisting of alpha (a) and beta (b) subunits, which form cell-to-matrix bonds, activate various pathways and thereby control cell proliferation, differentiation and survival.

Y- and A-TSPC were derived from human Achilles tendons and are fully described in Kohler et al. 2013. RT-PCR was used to assess the expression of collagen-binding integrins in the TSPC cultivated in collagen gels. Next, a1 and a11 integrins were silenced by stable lentiviral delivery of target-specific shRNA in the Y-TSPC. Control (con-shRNA), integrin (a1-shRNA) and integrin a11 (a11-shRNA) virus-containing supernatant was given for 24h and then cells were selected with 50 microg./ml zeocin for 10 days. The integrin knockdown (KD) efficiency was assessed by quantitative PCR and western blotting. Last, functional tests were carried out by time-lapse recording gel contraction of four cell groups (Y-TSPC+con, Y-TSPC+a1KD, Y-TSPC+a11KD, and A-TSPC).

Among the screened integrins we found that integrin a1 and a11 were significantly downregulated in A-TSPC with 3.8 and 5.6 folds, correspondingly. Therefore, to mimic the A-TSPC we carried out a gene KD of a1 and a11 in Y-TSPC. PCR and western blot clearly validated the efficient KD. Analyses of collagen contraction, revealed that Y-TSPC+a11KD significantly reduced collagen contractability comparable to A-TSPC. This indicated the indispensable role of this integrin in the signaling pathway of collagen matrix remodeling. In respect to integrin a1, we found that this receptor did not affect the contraction rate of Y-TSPC, which was similar to Y-TSPC+con.

To our knowledge we have now identified for the first time the critical role of a11 integrin receptor in tendon collagen remodeling, and a follow up analysis of its exact downstream cascade is on the way. Future efforts in deciphering how tendon matrix makeover is regulated can lead to innovation in preventive strategies for tendon degeneration.

Previous studies have shown that Tnmd is important for tendon maturation and has key implications for the residing tendon stem/progenitor cells. The putative signaling in which Tnmd participates is just starting to be better understood (Dex et al. 2016). However, its exact functions during tendon healing process still remain elusive. Therefore, the aims of this study were to perform systematic review of the literature on Tnmd-related research and to investigate the role of Tnmd in early tendon healing by applying a tendon rupture model in Tnmd-deficient mice.

First, we searched in the PubMed database for articles containing “tenomodulin” or its alternative names and abbreviations. After exclusion of papers only available in abstract form and foreign language, we grouped the remaining 128 full-text publications into four study types: 1) looking into functions of Tnmd; 2) using Tnmd as a tendon marker; 3) correlating Tnmd mutations to a variety of diseases; and 4) reviews. Following literature analysis, we carried out a pilot Achilles tendon injury model with Tnmd-knockout (KO) mouse strain. Adult Tnmd-KO (n = 8) and wild-type (WT) (n = 8) mice underwent unilateral surgery of Achilles tendon based on Palmes et al. 2002 and were compared at day 8 postoperatively by: 1) H&E staining for overall assessment; 2) immunohistochemical BrdU analysis for cell proliferation; and 3) Safranin O staining for endochondral formation.

Our literature screen revealed that Tnmd has been strongly justified as the best tendon and ligament marker in more than 90 different studies. Moreover, in vivo and in vitro investigations have demonstrated its positive role on tendon cell proliferation and tissue functions. Our follow up surgical study showed a very different scar organization in Tnmd-KO with a clearly reduced cell density. BrdU analysis confirmed a lower number of proliferating cells in Tnmd-KO scar area. Interestingly, endochondral formation was not observed in the scar tissues in either of the genotypes at day 8.

Taken together, we systematically summarized the current knowledge on Tnmd gene and highlighted several future research perspectives. Lack of studies on the role of Tnmd in tissue healing, motivated our pilot investigation on Achilles tendon rupture, which in turn suggested that loss of Tnmd results in inferior repair process.

Summary Statement

Progenitor cells from the periosteal niche are of great clinical interest due to their remarkable regenerative capacity. Here we report on progenitor cells from arthritic patients whose femoral neck periosteum was resected over the course of hip replacement.