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  • 1.
    Andersson, Gustav
    et al.
    Umeå University, Faculty of Medicine, Department of Integrative Medical Biology (IMB), Anatomy. Umeå University, Faculty of Medicine, Department of Surgical and Perioperative Sciences, Hand Surgery.
    Backman, Ludvig J.
    Umeå University, Faculty of Medicine, Department of Integrative Medical Biology (IMB), Anatomy.
    Christensen, Jens
    Umeå University, Faculty of Medicine, Department of Integrative Medical Biology (IMB), Anatomy.
    Alfredson, Håkan
    Umeå University, Faculty of Medicine, Department of Surgical and Perioperative Sciences, Sports Medicine.
    Nerve distributions in insertional Achilles tendinopathy - a comparison of bone, bursae and tendon2017In: Histology and Histopathology, ISSN 0213-3911, E-ISSN 1699-5848, Vol. 32, no 3, p. 263-270Article in journal (Refereed)
    Abstract [en]

    Background/Aim. In a condition of pain in the Achilles tendon insertion there are multiple structures involved, such as the Achilles tendon itself, the retrocalcaneal bursa and a bony protrusion at the calcaneal tuberosity called Haglund's deformity. The innervation patterns of these structures are scarcely described, and the subcutaneous calcaneal bursa is traditionally not considered to be involved in the pathology. This study aimed at describing the innervation patterns of the four structures described above to provide a better understanding of possible origins of pain at the Achilles tendon insertion.

    Methods. Biopsies were taken from 10 patients with insertional Achilles tendinopathy, which had pathological changes in the subcutaneous and retrocalcaneal bursae, a Haglund deformity and Achilles tendon tendinopathy as verified by ultrasound. The biopsies were stained using immunohistochemistry in order to delineate the innervation patterns in the structures involved in insertional Achilles tendinopathy.

    Results. Immunohistochemical examinations found that the subcutaneous bursa scored the highest using a semi-quantitative evaluation of the degree of innervation when compared to the retrocalcaneal bursa, the Achilles tendon, and the calcaneal bone.

    Conclusions. These findings suggest that the subcutaneous bursa, which is traditionally not included in surgical treatment, may be a clinically important factor in insertional Achilles tendinopathy.

  • 2.
    Andersson, Gustav
    et al.
    Umeå University, Faculty of Medicine, Department of Integrative Medical Biology (IMB), Anatomy.
    Backman, Ludvig
    Umeå University, Faculty of Medicine, Department of Integrative Medical Biology (IMB), Anatomy.
    Scott, Alexander
    Department of Physical Therapy, University of British Columbia, Vancouver, British Columbia, Canada; Centre for Hip Health and Mobility, Vancouver Coastal Health and Research Institute, Vancouver, British Columbia, Canada.
    Lorentzon, Ronny
    Umeå University, Faculty of Medicine, Department of Surgical and Perioperative Sciences, Sports Medicine.
    Forsgren, Sture
    Umeå University, Faculty of Medicine, Department of Integrative Medical Biology (IMB), Anatomy.
    Danielson, Patrik
    Umeå University, Faculty of Medicine, Department of Integrative Medical Biology (IMB), Anatomy.
    Substance P accelerates hypercellularity and angiogenesis in tendon tissue and enhances paratendinitis in response to Achilles tendon overuse in a tendinopathy model2011In: British Journal of Sports Medicine, ISSN 0306-3674, E-ISSN 1473-0480, Vol. 45, no 13, p. 1017-1022Article in journal (Refereed)
    Abstract [en]

    Background Tenocytes produce substance P (SP) and its receptor (neurokinin-1 receptor (NK-1R) is expressed throughout the tendon tissue, expecially in patients with tendinopathy and tissue changes (tendinosis) including hypercellularity and vascular proliferation. Considering the known effects of SP, one might ask whether SP contributes to these canges.

    Objectives To test whether development of tendinosislike changes (hypercellularity and angiogenesis) is accelerated during a 1-week course of ecercise with local administration of SP in an establish Achilles tendinopathy model.

    Methods Rabbits were subjected to a protocol of Achilles tendon overuse for 1 week, in conjunction with SP injections in the paratenon. Exercised control animals received NaCl injections or no injections, and unexercised, uninjected controls were also used. Tenocyte number and vascular density, as well as paratendinous inflammation, were evaluated. Immunohistochemistry and in sity hybridisation to detect NK-1R were conducted.

    Results There was a significant increase in tenocyte number in the SP-injected and NaCl-injected groups compared with both unexercised and exercised, uninjected controls. Tendon blood vessels increased in number in the SP-injected group compared with unexercised controls, a finding not seen in NaCl-injected controls or in uninjected, exercised animals. Paratendinous inflammation was more pronounced in the SP-injected group than in the NaCl controls. NK-1R was detected in blood vessel walls, nerves, inflammatory cells and tenocytes.

    Conclusions SP accelerated the development of tendinosis-like changes in the rabbit. Achilles tendon, which supports theories of a potential role of SP in tendinosis development; a fact of clinical interest since SP effects can be effectively blocked. The angiogenic response to SP injections seems related to parateninitis.

  • 3.
    Andersson, Gustav
    et al.
    Umeå University, Faculty of Medicine, Department of Integrative Medical Biology (IMB), Anatomy.
    Backman, Ludvig
    Umeå University, Faculty of Medicine, Department of Integrative Medical Biology (IMB), Anatomy.
    Scott, Alexander
    Department of Physical Therapy, University of British Columbia, Vancouver, BC, Canada.
    Lorentzon, Ronny
    Umeå University, Faculty of Medicine, Department of Surgical and Perioperative Sciences, Sports Medicine.
    Forsgren, Sture
    Umeå University, Faculty of Medicine, Department of Integrative Medical Biology (IMB), Anatomy.
    Danielson, Patrik
    Umeå University, Faculty of Medicine, Department of Integrative Medical Biology (IMB), Anatomy.
    Substance P induces tendinosis-like changes in a rabbit model of Achilles tendon overuseManuscript (preprint) (Other academic)
    Abstract [en]

    BACKGROUND: In previous studies we found evidence favouring that human Achilles tendon cells (tenocytes) are capable of producing the neuropeptide substance P (SP). Furthermore, the preferred receptor for SP (the neurokinin-1 receptor, NK-1 R) was widely expressed throughout the tendon, especially in patients suffering from chronic tendon pain (tendinopathy) with tissue changes (tendinosis) including hypercellularity and vascular proliferation. Considering known effects of SP, one might ask whether SP contributes to tendon cell proliferation and neovascularisation in tendinosis. We have an established animal (rabbit) model of Achilles tendinopathy based on overuse in the form of repetitive exercise. Recent studies with this model have shown that tendinosis-like changes are present after 3 weeks of exercise, but not after only 1 week. The current study aimed to test whether the development of tendinosis-like changes would be accelerated during a 1 week course of exercise with repetitive local administration of SP.

    MATERIAL AND METHODS: Four groups of animals (5-6 New Zealand white rabbits per group) were used. Three groups were subjected to the previously established protocol of Achilles tendon overuse for 1 week. One of these groups was given repetitive SP injections in the paratendinous tissue of the Achilles tendon, whereas one group (‘NaCl controls’) was given an equivalent schedule of saline injections. Two additional control groups existed: One in which the animals were neither subjected to the overuse protocol nor to any injections (‘untrained controls’), and one in which the animals trained for 1 week but were not given any injections (‘1 week controls’). Tenocyte number, vascular density, and the possible occurrence of paratendinous inflammation were evaluated. Immunohistochemistry and in situ hybridisation to detect NK-1 R were also conducted.

    RESULTS: There was a significant increase in tenocyte number in the SP-injected group compared to both untrained controls and 1 week controls. However, the same phenomenon was noticed for NaCl controls, i.e. tenocyte number was significantly increased in response to NaCl injections compared to untrained controls. There was an increase in the number of tendon blood vessels in the SP-injected group as compared to untrained controls, and this increase in vascularity was not seen for the NaCl controls or the 1 week controls. Paratendinous inflammation, as evidenced by invasion of inflammatory cells in the paratenon, was clearly more pronounced in the SP-injected group than in the NaCl controls. NK-1 R was detected in blood vessel walls, on nerves, on inflammatory cells, and on tenocytes.

    DISCUSSION AND CONCLUSIONS: The observations suggest that SP induces tenocyte proliferation and angiogenesis in the rabbit Achilles tendon, thus supporting a potential role of this neuropeptide in the processes that occur in tendinosis. The study corroborates findings on the human Achilles tendon in that NK-1 R was expressed on tenocytes and tendon blood vessel walls, thereby providing a potential anatomic basis for the observed effects of SP on the development of tendinosis. The hypercellularity observed in response to NaCl injections might be due increased tissue pressure or to stimulation of endogenous SPproduction, a phenomenon not unheard of. The angiogenic effect of SP injections, on the other hand, appeared to be more specifically related to an induction of inflammation in the paratendon.

  • 4.
    Backman, Ludvig
    Umeå University, Faculty of Medicine, Department of Integrative Medical Biology (IMB), Anatomy.
    Neuropeptide and catecholamine effects on tenocytes in tendinosis development: studies on two model systems with focus on proliferation and apoptosis2013Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    Background: Achilles tendinopathy is a common clinical syndrome of chronic Achilles tendon pain combined with thickening of the tendon and impaired tendon function. Tendinopathy is often, but not always, induced by mechanical overload, and is frequently accompanied by abnormalities at the tissue level, such as hypercellularity and angiogenesis, in which case the condition is called tendinosis. In tendinosis, there are no signs of intratendinous inflammation, but occasionally increased apoptosis is observed. Tendinosis is often hard to treat and its pathogenesis is still not clear. Recently, a new hypothesis has gained support, suggesting a biochemical model based on the presence of a non-neuronal production of classically neuronal signal substances by the primary tendon cells (tenocytes) in tendinosis. The possible functional importance of these signal substances in tendons is unknown and needs to be studied. In particular, the neuropeptide substance P (SP) and catecholamines are of interest in this regard, since these substances have been found to be up-regulated in tendinosis. As both SP and catecholamines are known to exert effects in other tissues resulting in changes similar to those characteristic of tendinosis, it is possible that they have a role in tendinosis development. It is furthermore unknown what elicits the increased intratendinous neuropeptide production in tendinosis, but given that tendon overload is a prominent riskfactor, it is possible that mechanical stimuli are involved.

    The hypothesis of this thesis work was that intratendinous production of SP is up-regulated in response to load of Achilles tendons/tenocytes, and thatstimulation of the preferred SP receptor, the neurokinin-1 receptor (NK-1 R), aswell as stimulation of the catecholamine α2 adrenoreceptors, contribute to the hypercellularity seen in tendinosis, via increased proliferation and/or decreased apoptosis, and that SP stimulates tendon angiogenesis. The purpose of the studies was to test this hypothesis. To achieve this, two model systems were used: One in vivo (rabbit Achilles tendon overload model of tendinosis) and one in vitro (human primary Achilles tendon cell culture model).

    Results: In the rabbit Achilles tendon tissue, SP and NK-1 R expression was extensive in the blood vessel walls, but also to some extent seen in the tenocytes. Quantification of endogenously produced SP in vivo confirmed intratendinous production of the peptide. The production of SP by human tendon cells in vitro was furthermore demonstrated. The catecholamine synthesizing enzyme tyrosine hydroxylase (TH), as well as the α2A adrenoreceptor (α2A AR), were detected in the tenocytes, both in vivo in the rabbit tissue and in vitro in the human tendon cells. As a response to mechanical loading in the in vivo model, the intratendinous levels of SP increased, and this elevation was found to precede distinct tendinosis changes. The in vitro model demonstrated the same response to load, i.e. an increased SP expression, but in this case also a decrease in the NK-1 R expression. In the in vivo model, exogenously administered SP, as well as clonidine (an α2 AR agonist), accelerated tenocyte hypercellularity, an effect that was not seen when administrating a specific α2A AR antagonist. Exogenous administration of SP also resulted in intratendinous angiogenesis and paratendinous inflammation. In the in vitro model, both SP and clonidine had proliferative effects on the human tenocytes, specifically mediated via NK-1R and α2A AR, respectively; both of which in turn involved activation/phosphorylation of the extracellular signal-regulated kinases 1 and 2 (ERK1/2). Exogenously administered SP, in Anti-Fas induced apoptosis of the tenocytes in vitro, confirmed SP to have an anti-apoptotic effect on these cells. This effect was specifically mediated via NK-1 R and the known anti-apoptotic Akt pathway.

    Conclusions: In summary, this thesis concludes that stimulation of NK-1 R and α2A AR on tenocytes, both in vitro and in vivo, mediates significant cell signalling effects leading to processes known to occur in tendinosis, including hypercellularity. The pathological role of the hypercellularity in tendinosis is still unclear, but it is likely to affect collagen metabolism/turnover and arrangement, and thereby indirectly tendon biomechanical function. Additional evidence is here provided showing that SP not only causes tenocyte proliferation, but also contributes to anti-apoptotic events. Furthermore, it was concluded that SP may be involved in the development of tendinosis, since its production is increased in response to load, preceding tendinosis, and since SP accelerates tendinosis changes, through some mechanistic pathways here delineated. These findings suggest that inhibition of SP, and possibly also catecholamines, could be beneficial in the reconstitution/normalization of tendon structure in tendinosis.

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  • 5.
    Backman, Ludvig
    et al.
    Umeå University, Faculty of Medicine, Department of Integrative Medical Biology (IMB), Anatomy. Umeå University, Faculty of Medicine, Department of Surgical and Perioperative Sciences, Sports Medicine.
    Andersson, Gustav
    Umeå University, Faculty of Medicine, Department of Integrative Medical Biology (IMB), Anatomy.
    Wennstig, Gabriel
    Umeå University, Faculty of Medicine, Department of Integrative Medical Biology (IMB), Anatomy.
    Forsgren, Sture
    Umeå University, Faculty of Medicine, Department of Integrative Medical Biology (IMB), Anatomy.
    Danielson, Patrik
    Umeå University, Faculty of Medicine, Department of Integrative Medical Biology (IMB), Anatomy.
    Endogenous substance P production in the Achilles tendon increases with loading in an in vivo model of tendinopathy: peptidergic elevation preceding tendinosis-like tissue changes2011In: Journal of Musculoskeletal and Neuronal Interactions - JMNI, ISSN 1108-7161, Vol. 11, no 2, p. 133-140Article in journal (Refereed)
    Abstract [en]

    Objectives: To quantify the intratendinous levels of substance P (SP) at different stages of overload in an established modelfor Achilles tendinopathy (rabbit). Also, to study the distribution of the SP-receptor, the NK-1R, and the source of SP, in thetendon. 

    Methods: Animals were subjected to the overuse protocol for 1, 3 or 6 weeks. One additional group served as unexercisedcontrols. Immunoassay (EIA), immunohistochemistry (IHC), and in situ hybridisation (ISH) were performed.

    Results: EIA revealedincreased SP-levels in the Achilles tendon of the exercised limb in all the experimental groups as compared to in thecontrols (statistically significant; p=0.01). A similar trend in the unexercised Achilles tendon was observed but was not statisticallysignificant (p=0.14). IHC and in ISH illustrated reactions of both SP and NK-1R mainly in blood vessel walls, but the receptorwas also found on tenocytes.

    Conclusions: Achilles tendon SP-levels are elevated already after 1 week of loading. This showsthat increased SP-production precedes tendinosis, as tendinosis-like changes occur only after a minimum of 3 weeks of exercise,as shown in a recent study using this model. We propose that central neuronal mechanism may be involved as similar trends wereobserved in the contralateral Achilles tendon.

  • 6.
    Backman, Ludvig
    et al.
    Umeå University, Faculty of Medicine, Department of Integrative Medical Biology (IMB), Anatomy. Umeå University, Faculty of Medicine, Department of Surgical and Perioperative Sciences, Sports Medicine.
    Fong, Gloria
    Umeå University, Faculty of Medicine, Department of Integrative Medical Biology (IMB), Anatomy.
    Andersson, Gustav
    Umeå University, Faculty of Medicine, Department of Integrative Medical Biology (IMB), Anatomy.
    Scott, Alexander
    Vancouver Coastal Health and Research Institute, University of British Columbia, Vancouver.
    Danielson, Patrik
    Umeå University, Faculty of Medicine, Department of Integrative Medical Biology (IMB), Anatomy.
    Substance P is a mechanoresponsive, autocrine regulator of human tenocyte proliferation2011In: PLOS ONE, E-ISSN 1932-6203, Vol. 6, no 11, p. e27209-Article in journal (Refereed)
    Abstract [en]

    It has been hypothesised that substance P (SP) may be produced by primary fibroblastic tendon cells (tenocytes), and that this production, together with the widespread distribution of the neurokinin-1 receptor (NK-1 R) in tendon tissue, could play an important role in the development of tendinopathy, a condition of chronic tendon pain and thickening. The aim of this study was to examine the possibility of endogenous SP production and the expression of NK-1 R by human tenocytes. Because tendinopathy is related to overload, and because the predominant tissue pathology (tendinosis) underlying early tendinopathy is characterized by tenocyte hypercellularity, the production of SP in response to loading/strain and the effects of exogenously administered SP on tenocyte proliferation were also studied. A cell culture model of primary human tendon cells was used. The vast majority of tendon cells were immunopositive for the tenocyte/fibroblast markers tenomodulin and vimentin, and immunocytochemical counterstaining revealed that positive immunoreactions for SP and NK-1 R were seen in a majority of these cells. Gene expression analyses showed that mechanical loading (strain) of tendon cell cultures using the FlexCell (R) technique significantly increased the mRNA levels of SP, whereas the expression of NK-1 R mRNA decreased in loaded as compared to unloaded tendon cells. Reduced NK-1 R protein was also observed, using Western blot, after exogenously administered SP at a concentration of 10(-7) M. SP exposure furthermore resulted in increased cell metabolism, increased cell viability, and increased cell proliferation, all of which were found to be specifically mediated via the NK-1 R; this in turn involving a common mitogenic cell signalling pathway, namely phosphorylation of ERK1/2. This study indicates that SP, produced by tenocytes in response to mechanical loading, may regulate proliferation through an autocrine loop involving the NK-1 R.

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  • 7.
    Backman, Ludvig J
    et al.
    Umeå University, Faculty of Medicine, Department of Integrative Medical Biology (IMB), Anatomy.
    Andersson, Gustav
    Umeå University, Faculty of Medicine, Department of Integrative Medical Biology (IMB), Anatomy.
    Fong, Gloria
    Umeå University, Faculty of Medicine, Department of Integrative Medical Biology (IMB), Anatomy. Department of Physical Therapy, University of British Columbia and Centre for Hip Health and Mobility, Vancouver Coastal Health and Research Institute, Vancouver, British Columbia, Canada.
    Alfredson, Håkan
    Umeå University, Faculty of Medicine, Department of Surgical and Perioperative Sciences, Sports Medicine.
    Scott, A
    University of British Columbia, Vancouver, Vancouver Coastal Health and Research Institute.
    Danielson, Patrik
    Umeå University, Faculty of Medicine, Department of Integrative Medical Biology (IMB), Anatomy.
    Alpha-2 adrenergic stimulation triggers Achilles tenocyte hypercellularity: comparison between two model systems2013In: Scandinavian Journal of Medicine and Science in Sports, ISSN 0905-7188, E-ISSN 1600-0838, Vol. 23, no 6, p. 687-696Article in journal (Refereed)
    Abstract [en]

    The histopathology of tendons with painful tendinopathy is often tendinosis, a fibrosis-like condition of unclear pathogenesis characterized by tissue changes including hypercellularity. The primary tendon cells (tenocytes) have been shown to express adrenoreceptors (mainly alpha-2A) as well as markers of catecholamine production, particularly in tendinosis. It is known that adrenergic stimulation can induce proliferation in other cells. The present study investigated the effects of an exogenously administered alpha-2 adrenergic agonist in an established in vivo Achilles tendinosis model (rabbit) and also in an in vitro human tendon cell culture model. The catecholamine producing enzyme tyrosine hydroxylase and the alpha-2A-adrenoreceptor (α(2A) AR) were expressed by tenocytes, and alpha-2 adrenergic stimulation had a proliferative effect on these cells, in both models. The proliferation was inhibited by administration of an α(2A) AR antagonist, and the in vitro model further showed that the proliferative alpha-2A effect was mediated via a mitogenic cell signaling pathway involving phosphorylation of extracellular-signal-regulated kinases 1 and 2. The results indicate that catecholamines produced by tenocytes in tendinosis might contribute to the proliferative nature of the pathology through stimulation of the α(2A) AR, pointing to a novel target for future therapies. The study furthermore shows that animal models are not necessarily required for all aspects of this research.

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  • 8.
    Backman, Ludvig J
    et al.
    Umeå University, Faculty of Medicine, Department of Integrative Medical Biology (IMB), Anatomy. Umeå University, Faculty of Medicine, Department of Surgical and Perioperative Sciences, Sports Medicine.
    Danielson, Patrik
    Umeå University, Faculty of Medicine, Department of Integrative Medical Biology (IMB), Anatomy.
    Akt-mediated anti-apoptotic effects of substance P in Anti-Fas-induced apoptosis of human tenocytes2013In: Journal of Cellular and Molecular Medicine (Print), ISSN 1582-1838, E-ISSN 1582-4934, Vol. 17, no 6, p. 723-733Article in journal (Refereed)
    Abstract [en]

    Substance P (SP) and its receptor, the neurokinin-1 receptor (NK-1 R), are expressed by human tenocytes, and they are both up-regulated incases of tendinosis, a condition associated with excessive apoptosis. It is known that SP can phosphorylate/activate the protein kinase Akt,which has anti-apoptotic effects. This mechanism has not been studied for tenocytes. The aims of this study were to investigate if Anti-Fastreatment is a good apoptosis model for human tenocytes in vitro, if SP protects from Anti-Fas-induced apoptosis, and by which mechanismsSP mediates an anti-apoptotic response. Anti-Fas treatment resulted in a time- and dose-dependent release of lactate dehydrogenase (LDH), i.e.induction of cell death, and SP dose-dependently reduced the Anti-Fas-induced cell death through a NK-1 R specific pathway. The same trendwas seen for the TUNEL assay, i.e. SP reduced Anti-Fas-induced apoptosis via NK-1 R. In addition, it was shown that SP reduces Anti-Fas-induced decrease in cell viability as shown with crystal violet assay. Protein analysis using Western blot confirmed that Anti-Fas inducescleavage/activation of caspase-3 and cleavage of PARP; both of which were inhibited by SP via NK-1 R. Finally, SP treatment resulted in phosphorylation/activation of Akt as shown with Western blot, and it was confirmed that the anti-apoptotic effect of SP was, at least partly, inducedthrough the Akt-dependent pathway. In conclusion, we show that SP reduces Anti-Fas-induced apoptosis in human tenocytes and that this antiapoptoticeffect of SP is mediated through NK-1 R and Akt-specific pathways.

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  • 9.
    Backman, Ludvig J
    et al.
    Umeå University, Faculty of Medicine, Department of Integrative Medical Biology (IMB), Anatomy.
    Danielson, Patrik
    Umeå University, Faculty of Medicine, Department of Integrative Medical Biology (IMB), Anatomy.
    Low range of ankle dorsiflexion predisposes for patellar tendinopathy in junior elite basketball players: a 1-year prospective study2011In: American Journal of Sports Medicine, ISSN 0363-5465, E-ISSN 1552-3365, Vol. 39, no 12, p. 2626-2633Article in journal (Refereed)
    Abstract [en]

    BACKGROUND: Patellar tendinopathy (PT) is one of the most common reasons for sport-induced pain of the knee. Low ankle dorsiflexion range might predispose for PT because of load-bearing compensation in the patellar tendon.

    PURPOSE: The purpose of this 1-year prospective study was to analyze if a low ankle dorsiflexion range increases the risk of developing PT for basketball players. STUDY DESIGN: Cohort study (prognosis); Level of evidence, 2.

    METHODS: Ninety junior elite basketball players were examined for different characteristics and potential risk factors for PT, including ankle dorsiflexion range in the dominant and nondominant leg. Data were collected over a 1-year period and follow-up, including reexamination, was made at the end of the year.

    RESULTS: Seventy-five players met the inclusion criteria. At the follow-up, 12 players (16.0%) had developed unilateral PT. These players were found to have had a significantly lower mean ankle dorsiflexion range at baseline than the healthy players, with a mean difference of -4.7° (P = .038) for the dominant limb and -5.1° (P = .024) for the nondominant limb. Complementary statistical analysis showed that players with dorsiflexion range less than 36.5° had a risk of 18.5% to 29.4% of developing PT within a year, as compared with 1.8% to 2.1% for players with dorsiflexion range greater than 36.5°. Limbs with a history of 2 or more ankle sprains had a slightly less mean ankle dorsiflexion range compared to those with 0 or 1 sprain (mean difference, -1.5° to -2.5°), although this was only statistically significant for nondominant legs.

    CONCLUSION: This study clearly shows that low ankle dorsiflexion range is a risk factor for developing PT in basketball players. In the studied material, an ankle dorsiflexion range of 36.5° was found to be the most appropriate cutoff point for prognostic screening. This might be useful information in identifying at-risk individuals in basketball teams and enabling preventive actions. A history of ankle sprains might contribute to reduced ankle dorsiflexion range.

  • 10.
    Backman, Ludvig J.
    et al.
    Umeå University, Faculty of Medicine, Department of Integrative Medical Biology (IMB), Anatomy. Umeå University, Faculty of Medicine, Department of Surgical and Perioperative Sciences, Sports Medicine.
    Eriksson, Daniella E.
    Umeå University, Faculty of Medicine, Department of Integrative Medical Biology (IMB), Anatomy.
    Danielson, Patrik
    Umeå University, Faculty of Medicine, Department of Integrative Medical Biology (IMB), Anatomy.
    Substance P reduces TNF-α-induced apoptosis in human tenocytes through NK-1 receptor stimulation2014In: British Journal of Sports Medicine, ISSN 0306-3674, E-ISSN 1473-0480, Vol. 48, no 19, p. 1414-1420Article in journal (Refereed)
    Abstract [en]

    BACKGROUND: It has been hypothesised that an upregulation of the neuropeptide substance P (SP) and its preferred receptor, the neurokinin-1 receptor (NK-1 R), is a causative factor in inducing tenocyte hypercellularity, a characteristic of tendinosis, through both proliferative and antiapoptotic stimuli. We have demonstrated earlier that SP stimulates proliferation of human tenocytes in culture.

    AIM: The aim of this study was to investigate whether SP can mediate an antiapoptotic effect in tumour necrosis factor-α (TNF-α)-induced apoptosis of human tenocytes in vitro.

    RESULTS: A majority (approximately 75%) of tenocytes in culture were immunopositive for TNF Receptor-1 and TNF Receptor-2. Exposure of the cells to TNF-α significantly decreased cell viability, as shown with crystal violet staining. TNF-α furthermore significantly increased the amount of caspase-10 and caspase-3 mRNA, as well as both BID and cleaved-poly ADP ribosome polymerase (c-PARP) protein. Incubation of SP together with TNF-α resulted in a decreased amount of BID and c-PARP, and in a reduced lactate dehydrogenase release, as compared to incubation with TNF-α alone. The SP effect was blocked with a NK-1 R inhibitor.

    DISCUSSION: This study shows that SP, through stimulation of the NK-1 R, has the ability to reduce TNF-α-induced apoptosis of human tenocytes. Considering that SP has previously been shown to stimulate tenocyte proliferation, the study confirms SP as a potent regulator of cell-turnover in tendon tissue, capable of stimulating hypercellularity through different mechanisms. This gives further support for the theory that the upregulated amount of SP seen in tendinosis could contribute to hypercellularity.

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  • 11.
    Borbely, Gabor
    et al.
    Umeå University, Faculty of Medicine, Department of Integrative Medical Biology (IMB).
    Löfgren, Filip
    Umeå University, Faculty of Medicine, Department of Integrative Medical Biology (IMB).
    Sloniecka, Marta
    Umeå University, Faculty of Medicine, Department of Integrative Medical Biology (IMB).
    Backman, Ludvig J.
    Umeå University, Faculty of Medicine, Department of Integrative Medical Biology (IMB).
    Danielson, Patrik
    Umeå University, Faculty of Medicine, Department of Integrative Medical Biology (IMB).
    The role of neurokinin A in corneal wound repair2015In: Investigative Ophthalmology and Visual Science, ISSN 0146-0404, E-ISSN 1552-5783, Vol. 56, no 7, article id Meeting Abstract: 725Article in journal (Other academic)
  • 12.
    Chen, Jialin
    et al.
    Umeå University, Faculty of Medicine, Department of Integrative Medical Biology (IMB), Anatomy. Department of Pathogenic Biology and Immunology, School of Medicine and Jiangsu Key Laboratory for Biomaterials and Devices, Southeast University, Nanjing, China.
    Backman, Ludvig J.
    Umeå University, Faculty of Medicine, Department of Integrative Medical Biology (IMB), Anatomy. Umeå University, Faculty of Medicine, Department of Community Medicine and Rehabilitation, Section of Physiotherapy.
    Zhang, Wei
    Umeå University, Faculty of Medicine, Department of Integrative Medical Biology (IMB), Anatomy. Jiangsu Key Laboratory for Biomaterials and Devices and Department of Physiology, School of Medicine, Southeast University, Nanjing, China.
    Ling, Chen
    Danielson, Patrik
    Umeå University, Faculty of Medicine, Department of Integrative Medical Biology (IMB), Anatomy. Umeå University, Faculty of Medicine, Department of Clinical Sciences, Ophthalmology.
    Regulation of Keratocyte Phenotype and Cell Behavior by Substrate Stiffness2020In: ACS Biomaterials Science & Engineering, E-ISSN 2373-9878, Vol. 6, no 9, p. 5162-5171Article in journal (Refereed)
    Abstract [en]

    Corneal tissue engineering is an alternative way to solve the problem of lack of corneal donor tissue in corneal transplantation. Keratocytes with a normal phenotype and function in tissue-engineered cornea would be critical for corneal regeneration. Although the role of extracellular/substrate material stiffness is well-known for the regulation of the cell phenotype and cell behavior in many different cell types, its effects in keratocyte culture have not yet been thoroughly studied. This project studied the effect of substrate stiffness on the keratocyte phenotype marker expression and typical cell behavior (cell adhesion, proliferation, and migration), and the possible mechanisms involved. Human primary keratocytes were cultured on tissue culture plastic (TCP, similar to 10(6) kPa) or on plates with the stiffness equivalent of physiological human corneal stroma (25 kPa) or vitreous body (1 kPa). The expression of keratocyte phenotype markers, cell adhesion, proliferation, and migration were compared. The results showed that the stiffness of the substrate material regulates the phenotype marker expression and cell behavior of cultured keratocytes. Physiological corneal stiffness (25 kPa) superiorly preserved the cell phenotype when compared to the TCP and 1 kPa group. Keratocytes had a larger cell area when cultured on 25 kPa plates as compared to on TCP. Treatment of cells with NSC 23766 (Rac1 inhibitor) mimicked the response in the cell phenotype and behavior seen in the transition from soft materials to stiff materials, including the cytoskeletal structure, expression of keratocyte phenotype markers, and cell behavior. In conclusion, this study shows that substrate stiffness regulates the cell phenotype marker expression and cell behavior of keratocytes by Rac1-mediated cytoskeletal reorganization. This knowledge contributes to the development of corneal tissue engineering.

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  • 13.
    Chen, Jialin
    et al.
    Umeå University, Faculty of Medicine, Department of Integrative Medical Biology (IMB), Anatomy.
    Chen, Peng
    Backman, Ludvig J.
    Umeå University, Faculty of Medicine, Department of Integrative Medical Biology (IMB), Anatomy.
    Zhou, Qingjun
    Danielson, Patrik
    Umeå University, Faculty of Medicine, Department of Integrative Medical Biology (IMB), Anatomy. Umeå University, Faculty of Medicine, Department of Clinical Sciences, Ophthalmology.
    Ciliary Neurotrophic Factor Promotes the Migration of Corneal Epithelial Stem/progenitor Cells by Up-regulation of MMPs through the Phosphorylation of Akt2016In: Scientific Reports, E-ISSN 2045-2322, Vol. 6, article id 25870Article in journal (Refereed)
    Abstract [en]

    The migration of limbal epithelial stem cells is important for the homeostasis and regeneration of corneal epithelium. Ciliary neurotrophic factor (CNTF) has been found to promote corneal epithelial wound healing by activating corneal epithelial stem/progenitor cells. However, the possible effect of CNTF on the migration of corneal epithelial stem/progenitor cells is not clear. This study found the expression of CNTF in mouse corneal epithelial stem/progenitor cells (TKE2) to be up-regulated after injury, on both gene and protein level. CNTF promoted migration of TKE2 in a dose-dependent manner and the peak was seen at 10 ng/ml. The phosphorylation level of Akt (p-Akt), and the expression of MMP3 and MMP14, were up-regulated after CNTF treatment both in vitro and in vivo. Akt and MMP3 inhibitor treatment delayed the migration effect by CNTF. Finally, a decreased expression of MMP3 and MMP14 was observed when Akt inhibitor was applied both in vitro and in vivo. This study provides new insights into the role of CNTF on the migration of corneal epithelial stem/progenitor cells and its inherent mechanism of Up-regulation of matrix metalloproteinases through the Akt signalling pathway.

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  • 14.
    Chen, Jialin
    et al.
    Umeå University, Faculty of Medicine, Department of Integrative Medical Biology (IMB), Anatomy.
    Lan, Jie
    Liu, Dongle
    Backman, Ludvig J.
    Umeå University, Faculty of Medicine, Department of Integrative Medical Biology (IMB), Anatomy.
    Zhang, Wei
    Umeå University, Faculty of Medicine, Department of Integrative Medical Biology (IMB), Anatomy.
    Zhou, Qingjun
    Danielson, Patrik
    Umeå University, Faculty of Medicine, Department of Integrative Medical Biology (IMB), Anatomy.
    Ascorbic Acid Promotes the Stemness of Corneal Epithelial Stem/Progenitor Cells and Accelerates Epithelial Wound Healing in the Cornea2017In: Stem Cells Translational Medicine, ISSN 2157-6564, E-ISSN 2157-6580, Vol. 6, no 5, p. 1356-1365Article in journal (Refereed)
    Abstract [en]

    High concentration of ascorbic acid (vitamin C) has been found in corneal epithelium of various species. However, the specific functions and mechanisms of ascorbic acid in the repair of corneal epithelium are not clear. In this study, it was found that ascorbic acid accelerates corneal epithelial wound healing in vivo in mouse. In addition, ascorbic acid enhanced the stemness of cultured mouse corneal epithelial stem/progenitor cells (TKE2) in vitro, as shown by elevated clone formation ability and increased expression of stemness markers (especially p63 and SOX2). The contribution of ascorbic acid on the stemness enhancement was not dependent on the promotion of Akt phosphorylation, as concluded by using Akt inhibitor, nor was the stemness found to be dependent on the regulation of oxidative stress, as seen by the use of two other antioxidants (GMEE and NAC). However, ascorbic acid was found to promote extracellular matrix (ECM) production, and by using two collagen synthesis inhibitors (AzC and CIS), the increased expression of p63 and SOX2 by ascorbic acid was decreased by around 50%, showing that the increased stemness by ascorbic acid can be attributed to its regulation of ECM components. Moreover, the expression of p63 and SOX2 was elevated when TKE2 cells were cultured on collagen I coated plates, a situation that mimics the in vivo situation as collagen I is the main component in the corneal stroma. This study shows direct therapeutic benefits of ascorbic acid on corneal epithelial wound healing and provides new insights into the mechanisms involved.

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  • 15.
    Chen, Jialin
    et al.
    School of Medicine, Southeast University, Nanjing, China; Center for Stem Cell and Regenerative Medicine, Southeast University, Nanjing, China; Jiangsu Key Laboratory for Biomaterials and Devices, Southeast University, Nanjing, China; China Orthopedic Regenerative Medicine Group (CORMed), Hangzhou, China.
    Mo, Qingyun
    School of Medicine, Southeast University, Nanjing, China; Center for Stem Cell and Regenerative Medicine, Southeast University, Nanjing, China.
    Long, Qiuzi
    Center for Stem Cell and Regenerative Medicine, Southeast University, Nanjing, China; Nanjing University of Chinese Medicine, Nanjing, China.
    Sheng, Renwang
    School of Medicine, Southeast University, Nanjing, China; Center for Stem Cell and Regenerative Medicine, Southeast University, Nanjing, China.
    Chen, Zhixuan
    School of Medicine, Southeast University, Nanjing, China; Center for Stem Cell and Regenerative Medicine, Southeast University, Nanjing, China.
    Luo, Yifan
    School of Medicine, Southeast University, Nanjing, China; Center for Stem Cell and Regenerative Medicine, Southeast University, Nanjing, China.
    Liu, Chuanquan
    School of Medicine, Southeast University, Nanjing, China; Center for Stem Cell and Regenerative Medicine, Southeast University, Nanjing, China.
    Backman, Ludvig J.
    Umeå University, Faculty of Medicine, Department of Integrative Medical Biology (IMB), Anatomy. Umeå University, Faculty of Medicine, Department of Community Medicine and Rehabilitation, Section of Physiotherapy.
    Zhang, Yanan
    School of Medicine, Southeast University, Nanjing, China; Center for Stem Cell and Regenerative Medicine, Southeast University, Nanjing, China.
    Zhang, Wei
    School of Medicine, Southeast University, Nanjing, China; Center for Stem Cell and Regenerative Medicine, Southeast University, Nanjing, China; Jiangsu Key Laboratory for Biomaterials and Devices, Southeast University, Nanjing, China; China Orthopedic Regenerative Medicine Group (CORMed), Hangzhou, China.
    Hydroxycamptothecin and substratum stiffness synergistically regulate fibrosis of human corneal fibroblasts2023In: ACS Biomaterials Science & Engineering, E-ISSN 2373-9878, Vol. 9, no 2, p. 959-967Article in journal (Refereed)
    Abstract [en]

    Corneal fibrosis is a common outcome of inappropriate repair associated with trauma or ocular infection. Altered biomechanical properties with increased corneal stiffness is a feature of fibrosis that cause corneal opacities, resulting in severe visual impairment and even blindness. The present study aims to determine the effect of hydroxycamptothecin (HCPT) and matrix stiffness on transforming growth factor-β1 (TGF-β1)-induced fibrotic processes in human corneal fibroblasts (HTK cells). HTK cells were cultured on substrates with different stiffnesses ("soft", ∼261 kPa; "stiff", ∼2.5 × 103 kPa) and on tissue culture plastic (TCP, ∼106 kPa) and simultaneously treated with or without 1 μg/mL HCPT and 10 ng/mL TGF-β1. We found that HCPT induced decreased cell viability and antiproliferative effects on HTK cells. TGF-β1-induced expression of fibrosis-related genes (FN1, ACTA2) was reduced if the cells were simultaneously treated with HCPT. Substrate stiffness did not affect the expression of fibrosis-related genes. The TGF-β1 induced expression of FN1 on both soft and stiff substrates was reduced if cells were simultaneously treated with HCPT. However, this trend was not seen for ACTA2, i.e., the TGF-β1 induced expression of ACTA2 was not reduced by simultaneous treatment of HCPT in either soft or stiff substrate. Instead, HCPT treatment in the presence of TGF-β1 resulted in increased gene expression of keratocyte phenotype makers (LUM, KERA, AQP1, CHTS6) on both substrate stiffnesses. In addition, the protein expression of keratocyte phenotype makers LUM and ALDH3 was increased in HTK cells simultaneously treated with TGF-β1 and HCPT on stiff substrate as compared to control, i.e., without HCPT. In conclusion, we found that HCPT can reduce TGF-β1-induced fibrosis and promote the keratocyte phenotype in a substrate stiffness dependent manner. Thus, HCPT stimulation might be an approach to stimulate keratocytes in the appropriate healing stage to avoid or reverse fibrosis and achieve more optimal corneal wound healing.

  • 16.
    Chen, Jialin
    et al.
    Umeå University, Faculty of Medicine, Department of Integrative Medical Biology (IMB), Anatomy. Center for Stem Cell and Tissue Engineering, School of Medicine, Zhejiang University, Hangzhou, Zhejiang; Zhejiang Provincial Key Lab forTissue Engineering and Regenerative Medicine, Hangzhou, Zhejiang, People’s Republic of China.
    Zhang, Erchen
    Zhang, Wei
    Liu, Zeyu
    Lu, Ping
    Zhu, Ting
    Yin, Zi
    Backman, Ludvig J.
    Umeå University, Faculty of Medicine, Department of Integrative Medical Biology (IMB), Anatomy.
    Liu, Huanhuan
    Chen, Xiao
    Ouyang, Hongwei
    Fos Promotes Early Stage Teno-Lineage Differentiation of Tendon Stem/Progenitor Cells in Tendon2017In: Stem Cells Translational Medicine, ISSN 2157-6564, E-ISSN 2157-6580, Vol. 6, no 11, p. 2009-2019Article in journal (Refereed)
    Abstract [en]

    Stem cells have been widely used in tendon tissue engineering. The lack of refined and controlled differentiation strategy hampers the tendon repair and regeneration. This study aimed to find new effective differentiation factors for stepwise tenogenic differentiation. By microarray screening, the transcript factor Fos was found to be expressed in significantly higher amounts in postnatal Achilles tendon tissue derived from 1 day as compared with 7-days-old rats. It was further confirmed that expression of Fos decreased with time in postnatal rat Achilles tendon, which was accompanied with the decreased expression of multiply tendon markers. The expression of Fos also declined during regular in vitro cell culture, which corresponded to the loss of tendon phenotype. In a cell-sheet and a three-dimensional cell culture model, the expression of Fos was upregulated as compared with in regular cell culture, together with the recovery of tendon phenotype. In addition, significant higher expression of tendon markers was found in Fos-overexpressed tendon stem/progenitor cells (TSPCs), and Fos knock-down gave opposite results. In situ rat tendon repair experiments found more normal tendon-like tissue formed and higher tendon markers expression at 4 weeks postimplantation of Fos-overexpressed TSPCs derived nonscaffold engineering tendon (cell-sheet), as compared with the control group. This study identifies Fos as a new marker and functional driver in the early stage teno-lineage differentiation of tendon, which paves the way for effective stepwise tendon differentiation and future tendon regeneration.

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  • 17.
    Chen, Jialin
    et al.
    Umeå University, Faculty of Medicine, Department of Integrative Medical Biology (IMB).
    Zhang, Wei
    Umeå University, Faculty of Medicine, Department of Integrative Medical Biology (IMB).
    Backman, Ludvig J.
    Umeå University, Faculty of Medicine, Department of Integrative Medical Biology (IMB).
    Kelk, Peyman
    Umeå University, Faculty of Medicine, Department of Integrative Medical Biology (IMB).
    Danielson, Patrik
    Umeå University, Faculty of Medicine, Department of Integrative Medical Biology (IMB). Umeå University, Faculty of Medicine, Department of Clinical Sciences, Ophthalmology.
    Mechanical stress potentiates the differentiation of periodontal ligament stem cells into keratocytes2018In: British Journal of Ophthalmology, ISSN 0007-1161, E-ISSN 1468-2079, Vol. 102, no 4, p. 562-569Article in journal (Refereed)
    Abstract [en]

    Aims To explore the role of corneal-shaped static mechanical strain on the differentiation of human periodontal ligament stem cells (PDLSCs) into keratocytes and the possible synergistic effects of mechanics and inducing medium. Methods PDLSCs were exposed to 3% static dome-shaped mechanical strain in a Flexcell Tension System for 3 days and 7 days. Keratocyte phenotype was determined by gene expression of keratocyte markers. Keratocyte differentiation (inducing) medium was introduced in the Flexcell system, either continuously or intermittently combined with mechanical stimulation. The synergistic effects of mechanics and inducing medium on keratocyte differentiation was evaluated by gene and protein expression of keratocyte markers. Finally, a multilamellar cell sheet was assembled by seeding PDLSCs on a collagen membrane and inducing keratocyte differentiation. The transparency of the cell sheet was assessed, and typical markers of native human corneal stroma were evaluated by immunofluorescence staining. Results Dome-shaped mechanical stimulation promoted PDLSCs to differentiate into keratocytes, as shown by the upregulation of ALDH3A1, CD34, LUM, COL I and COL V. The expression of integrins were also upregulated after mechanical stimulation, including integrin alpha 1, alpha 2, beta 1 and non-muscle myosin II B. A synergistic effect of mechanics and inducing medium was found on keratocyte differentiation. The cell sheets were assembled under the treatment of mechanics and inducing medium simultaneously. The cell sheets were transparent, multilamellar and expressed typical markers of corneal stroma. Conclusion Dome-shaped mechanical stimulation promotes differentiation of PDLSCs into keratocytes and has synergistic effects with inducing medium. Multilamellar cell sheets that resemble native human corneal stroma show potential for future clinical applications.

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  • 18.
    Chen, Jialin
    et al.
    Umeå University, Faculty of Medicine, Department of Integrative Medical Biology (IMB), Anatomy.
    Zhang, Wei
    Umeå University, Faculty of Medicine, Department of Integrative Medical Biology (IMB), Anatomy.
    Kelk, Peyman
    Umeå University, Faculty of Medicine, Department of Integrative Medical Biology (IMB), Anatomy.
    Backman, Ludvig J.
    Umeå University, Faculty of Medicine, Department of Integrative Medical Biology (IMB), Anatomy. Umeå University, Faculty of Medicine, Department of Community Medicine and Rehabilitation, Physiotherapy.
    Danielson, Patrik
    Umeå University, Faculty of Medicine, Department of Integrative Medical Biology (IMB), Anatomy. Umeå University, Faculty of Medicine, Department of Clinical Sciences, Ophthalmology.
    Substance P and patterned silk biomaterial stimulate periodontal ligament stem cells to form corneal stroma in a bioengineered three-dimensional model2017In: Stem Cell Research & Therapy, E-ISSN 1757-6512, Vol. 8, article id 260Article in journal (Refereed)
    Abstract [en]

    Background: We aimed to generate a bioengineered multi-lamellar human corneal stroma tissue in vitro by differentiating periodontal ligament stem cells (PDLSCs) towards keratocytes on an aligned silk membrane.

    Methods: Human PDLSCs were isolated and identified. The neuropeptide substance P (SP) was added in keratocyte differentiation medium (KDM) to evaluate its effect on keratocyte differentiation of PDLSCs. PDLSCs were then seeded on patterned silk membrane and cultured with KDM and SP. Cell alignment was evaluated and the expression of extracellular matrix (ECM) components of corneal stroma was detected. Finally, multi-lamellar tissue was constructed in vitro by PDLSCs seeded on patterned silk membranes, which were stacked orthogonally and stimulated by KDM supplemented with SP for 18 days. Sections were prepared and subsequently stained with hematoxylin and eosin or antibodies for immunofluorescence observation of human corneal stroma-related proteins.

    Results: SP promoted the expression of corneal stroma-related collagens (collagen types I, III, V, and VI) during the differentiation induced by KDM. Patterned silk membrane guided cell alignment of PDLSCs, and important ECM components of the corneal stroma were shown to be deposited by the cells. The constructed multi-lamellar tissue was found to support cells growing between every two layers and expressing the main type of collagens (collagen types I and V) and proteoglycans (lumican and keratocan) of normal human corneal stroma.

    Conclusions: Multi-lamellar human corneal stroma-like tissue can be constructed successfully in vitro by PDLSCs seeded on orthogonally aligned, multi-layered silk membranes with SP supplementation, which shows potential for future corneal tissue engineering.

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  • 19.
    Chen, Jialin
    et al.
    Umeå University, Faculty of Medicine, Department of Integrative Medical Biology (IMB), Anatomy.
    Zhang, Wei
    Liu, Zeyu
    Zhu, Ting
    Shen, Weiliang
    Ran, Jisheng
    Tang, Qiaomei
    Gong, Xiaonan
    Backman, Ludvig J.
    Umeå University, Faculty of Medicine, Department of Integrative Medical Biology (IMB), Anatomy.
    Chen, Xiao
    Chen, Xiaowen
    Wen, Feiqiu
    Ouyang, Hongwei
    Characterization and comparison of post-natal rat Achilles tendon-derived stem cells at different development stages2016In: Scientific Reports, E-ISSN 2045-2322, Vol. 6, article id 22946Article in journal (Refereed)
    Abstract [en]

    Tendon stem/progenitor cells (TSPCs) are a potential cell source for tendon tissue engineering. The striking morphological and structural changes of tendon tissue during development indicate the complexity of TSPCs at different stages. This study aims to characterize and compare post-natal rat Achilles tendon tissue and TSPCs at different stages of development. The tendon tissue showed distinct differences during development: the tissue structure became denser and more regular, the nuclei became spindle-shaped and the cell number decreased with time. TSPCs derived from 7 day Achilles tendon tissue showed the highest self-renewal ability, cell proliferation, and differentiation potential towards mesenchymal lineage, compared to TSPCs derived from 1 day and 56 day tissue. Microarray data showed up-regulation of several groups of genes in TSPCs derived from 7 day Achilles tendon tissue, which may account for the unique cell characteristics during this specific stage of development. Our results indicate that TSPCs derived from 7 day Achilles tendon tissue is a superior cell source as compared to TSPCs derived from 1 day and 56 day tissue, demonstrating the importance of choosing a suitable stem cell source for effective tendon tissue engineering and regeneration.

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  • 20.
    Chen, Zhixuan
    et al.
    School of Medicine, Southeast University, Nanjing, China; Center for Stem Cell and Regenerative Medicine, Southeast University, Nanjing, China.
    Zhang, Wei
    School of Medicine, Southeast University, Nanjing, China; Center for Stem Cell and Regenerative Medicine, Southeast University, Nanjing, China; Jiangsu Key Laboratory for Biomaterials and Devices, Southeast University, Nanjing, China; China Orthopedic Regenerative Medicine Group (CORMed), Hangzhou, China.
    Wang, Mingyue
    School of Medicine, Southeast University, Nanjing, China; Center for Stem Cell and Regenerative Medicine, Southeast University, Nanjing, China.
    Backman, Ludvig J.
    Umeå University, Faculty of Medicine, Department of Integrative Medical Biology (IMB), Anatomy. Umeå University, Faculty of Medicine, Department of Community Medicine and Rehabilitation, Section of Physiotherapy.
    Chen, Jialin
    School of Medicine, Southeast University, Nanjing, China; Center for Stem Cell and Regenerative Medicine, Southeast University, Nanjing, China; Jiangsu Key Laboratory for Biomaterials and Devices, Southeast University, Nanjing, China; China Orthopedic Regenerative Medicine Group (CORMed), Hangzhou, China.
    Effects of Zinc, Magnesium, and Iron Ions on Bone Tissue Engineering2022In: ACS Biomaterials Science & Engineering, E-ISSN 2373-9878, Vol. 8, no 6, p. 2321-2335Article, review/survey (Refereed)
    Abstract [en]

    Large-sized bone defects are a great challenge in clinics and considerably impair the quality of patients' daily life. Tissue engineering strategies using cells, scaffolds, and bioactive molecules to regulate the microenvironment in bone regeneration is a promising approach. Zinc, magnesium, and iron ions are natural elements in bone tissue and participate in many physiological processes of bone metabolism and therefore have great potential for bone tissue engineering and regeneration. In this review, we performed a systematic analysis on the effects of zinc, magnesium, and iron ions in bone tissue engineering. We focus on the role of these ions in properties of scaffolds (mechanical strength, degradation, osteogenesis, antibacterial properties, etc.). We hope that our summary of the current research achievements and our notifications of potential strategies to improve the effects of zinc, magnesium, and iron ions in scaffolds for bone repair and regeneration will find new inspiration and breakthroughs to inspire future research.

  • 21.
    Chi, Jiayu
    et al.
    School of Medicine, Southeast University, Nanjing, China.
    Wang, Mingyue
    School of Medicine, Southeast University, Nanjing, China.
    Chen, Jialin
    School of Medicine, Southeast University, Nanjing, China; Jiangsu Key Laboratory for Biomaterials and Devices, Southeast University, Nanjing, China; China Orthopedic Regenerative Medicine Group (CORMed), Hangzhou, China.
    Hu, Lizhi
    School of Medicine, Southeast University, Nanjing, China.
    Chen, Zhixuan
    School of Medicine, Southeast University, Nanjing, China.
    Backman, Ludvig J.
    Umeå University, Faculty of Medicine, Department of Integrative Medical Biology (IMB). Umeå University, Faculty of Medicine, Department of Community Medicine and Rehabilitation, Section of Physiotherapy.
    Zhang, Wei
    School of Medicine, Southeast University, Nanjing, China; Jiangsu Key Laboratory for Biomaterials and Devices, Southeast University, Nanjing, China; China Orthopedic Regenerative Medicine Group (CORMed), Hangzhou, China.
    Topographic Orientation of Scaffolds for Tissue Regeneration: Recent Advances in Biomaterial Design and Applications2022In: Biomimetics, E-ISSN 2313-7673, Vol. 7, no 3, article id 131Article, review/survey (Refereed)
    Abstract [en]

    Tissue engineering to develop alternatives for the maintenance, restoration, or enhancement of injured tissues and organs is gaining more and more attention. In tissue engineering, the scaffold used is one of the most critical elements. Its characteristics are expected to mimic the native extracellular matrix and its unique topographical structures. Recently, the topographies of scaffolds have received increasing attention, not least because different topographies, such as aligned and random, have different repair effects on various tissues. In this review, we have focused on various technologies (electrospinning, directional freeze-drying, magnetic freeze-casting, etching, and 3-D printing) to fabricate scaffolds with different topographic orientations, as well as discussed the physicochemical (mechanical properties, porosity, hydrophilicity, and degradation) and biological properties (morphology, distribution, adhesion, proliferation, and migration) of different topographies. Subsequently, we have compiled the effect of scaffold orientation on the regeneration of vessels, skin, neural tissue, bone, articular cartilage, ligaments, tendons, cardiac tissue, corneas, skeletal muscle, and smooth muscle. The compiled information in this review will facilitate the future development of optimal topographical scaffolds for the regeneration of certain tissues. In the majority of tissues, aligned scaffolds are more suitable than random scaffolds for tissue repair and regeneration. The underlying mechanism explaining the various effects of aligned and random orientation might be the differences in “contact guidance”, which stimulate certain biological responses in cells.

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  • 22.
    Dennhag, Nils
    et al.
    Umeå University, Faculty of Medicine, Department of Integrative Medical Biology (IMB). Umeå University, Faculty of Medicine, Department of Clinical Sciences.
    Kahsay, Abraha
    Umeå University, Faculty of Medicine, Department of Integrative Medical Biology (IMB).
    Nissen, Itzel
    Umeå University, Faculty of Medicine, Umeå Centre for Molecular Medicine (UCMM). Umeå University, Faculty of Medicine, Wallenberg Centre for Molecular Medicine at Umeå University (WCMM).
    Chermenina, Maria
    Umeå University, Faculty of Medicine, Department of Clinical Sciences.
    Nord, Hanna
    Umeå University, Faculty of Medicine, Department of Integrative Medical Biology (IMB).
    Liu, Jiao
    Arner, Anders
    Liu, Jing-Xia
    Umeå University, Faculty of Medicine, Department of Integrative Medical Biology (IMB).
    Backman, Ludvig J.
    Umeå University, Faculty of Medicine, Department of Integrative Medical Biology (IMB).
    Remeseiro, Silvia
    Umeå University, Faculty of Medicine, Umeå Centre for Molecular Medicine (UCMM). Umeå University, Faculty of Medicine, Wallenberg Centre for Molecular Medicine at Umeå University (WCMM).
    von Hofsten, Jonas
    Umeå University, Faculty of Medicine, Department of Integrative Medical Biology (IMB).
    Domellöf, Fatima Pedrosa
    Umeå University, Faculty of Medicine, Department of Integrative Medical Biology (IMB). Umeå University, Faculty of Medicine, Department of Clinical Sciences. Umeå University, Faculty of Medicine, Department of Medical Biosciences.
    fhl2b expression ameliorates muscular dystrophyManuscript (preprint) (Other academic)
  • 23.
    Dennhag, Nils
    et al.
    Umeå University, Faculty of Medicine, Department of Clinical Sciences, Ophthalmology. Umeå University, Faculty of Medicine, Department of Medical and Translational Biology.
    Kahsay, Abraha
    Umeå University, Faculty of Medicine, Department of Medical and Translational Biology. Umeå University, Faculty of Medicine, Department of Clinical Sciences, Ophthalmology.
    Nissen, Itzel
    Umeå University, Faculty of Medicine, Wallenberg Centre for Molecular Medicine at Umeå University (WCMM). Umeå University, Faculty of Medicine, Department of Medical and Translational Biology.
    Nord, Hanna
    Umeå University, Faculty of Medicine, Department of Medical and Translational Biology.
    Chermenina, Maria
    Umeå University, Faculty of Medicine, Department of Medical and Translational Biology. Umeå University, Faculty of Medicine, Department of Clinical Sciences, Ophthalmology.
    Liu, Jiao
    Div. Thoracic Surgery, Dept. Clinical Sciences, Lund University, Lund, Sweden; College of Life Sciences, South-Central University for Nationalities, Wuhan, China.
    Arner, Anders
    Div. Thoracic Surgery, Dept. Clinical Sciences, Lund University, Lund, Sweden.
    Liu, Jing-Xia
    Umeå University, Faculty of Medicine, Department of Medical and Translational Biology.
    Backman, Ludvig J.
    Umeå University, Faculty of Medicine, Department of Medical and Translational Biology.
    Remeseiro, Silvia
    Umeå University, Faculty of Medicine, Wallenberg Centre for Molecular Medicine at Umeå University (WCMM). Umeå University, Faculty of Medicine, Department of Medical and Translational Biology.
    von Hofsten, Jonas
    Umeå University, Faculty of Medicine, Department of Medical and Translational Biology.
    Domellöf, Fatima Pedrosa
    Umeå University, Faculty of Medicine, Department of Clinical Sciences, Ophthalmology. Umeå University, Faculty of Medicine, Department of Medical and Translational Biology.
    fhl2b mediates extraocular muscle protection in zebrafish models of muscular dystrophies and its ectopic expression ameliorates affected body muscles2024In: Nature Communications, E-ISSN 2041-1723, Vol. 15, no 1, article id 1950Article in journal (Refereed)
    Abstract [en]

    In muscular dystrophies, muscle fibers loose integrity and die, causing significant suffering and premature death. Strikingly, the extraocular muscles (EOMs) are spared, functioning well despite the disease progression. Although EOMs have been shown to differ from body musculature, the mechanisms underlying this inherent resistance to muscle dystrophies remain unknown. Here, we demonstrate important differences in gene expression as a response to muscle dystrophies between the EOMs and trunk muscles in zebrafish via transcriptomic profiling. We show that the LIM-protein Fhl2 is increased in response to the knockout of desmin, plectin and obscurin, cytoskeletal proteins whose knockout causes different muscle dystrophies, and contributes to disease protection of the EOMs. Moreover, we show that ectopic expression of fhl2b can partially rescue the muscle phenotype in the zebrafish Duchenne muscular dystrophy model sapje, significantly improving their survival. Therefore, Fhl2 is a protective agent and a candidate target gene for therapy of muscular dystrophies.

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  • 24.
    El-Habta, Roine
    et al.
    Umeå University, Faculty of Medicine, Department of Integrative Medical Biology (IMB), Anatomy.
    Andersson, Gustav
    Umeå University, Faculty of Medicine, Department of Integrative Medical Biology (IMB), Anatomy. Umeå University, Faculty of Medicine, Department of Surgical and Perioperative Sciences, Hand Surgery.
    Kingham, Paul J.
    Umeå University, Faculty of Medicine, Department of Integrative Medical Biology (IMB), Anatomy.
    Backman, Ludvig J.
    Umeå University, Faculty of Medicine, Department of Integrative Medical Biology (IMB), Anatomy.
    Anti-apoptotic effect of adipose tissue-derived stromal vascular fraction in denervated rat muscle2021In: Stem Cell Research & Therapy, E-ISSN 1757-6512, Vol. 12, no 162Article in journal (Refereed)
  • 25.
    El-Habta, Roine
    et al.
    Umeå University, Faculty of Medicine, Department of Integrative Medical Biology (IMB), Anatomy.
    Chen, Jialin
    Umeå University, Faculty of Medicine, Department of Integrative Medical Biology (IMB), Anatomy.
    Pingel, Jessica
    Backman, Ludvig J.
    Umeå University, Faculty of Medicine, Department of Integrative Medical Biology (IMB), Anatomy. Umeå University, Faculty of Medicine, Department of Community Medicine and Rehabilitation, Physiotherapy.
    Tendinosis-like changes in denervated rat Achilles tendon2018In: BMC Musculoskeletal Disorders, E-ISSN 1471-2474, Vol. 19, article id 426Article in journal (Refereed)
    Abstract [en]

    Background: Tendon disorders are common and lead to significant disability and pain. Our knowledge of the ‘tennis elbow’, the ‘jumpers knee’, and Achilles tendinosis has increased over the years, but changes in denervated tendons is yet to be described in detail. The aim of the present study was to investigate the morphological and biochemical changes in tendon tissue following two weeks of denervation using a unilateral sciatic nerve transection model in rat Achilles tendons.

    Methods: Tendons were compared with respect to cell number, nuclear roundness, and fiber structure. The non-denervated contralateral tendon served as a control. Also, the expression of neuromodulators such as substance P and its preferred receptor neurokinin-1 receptor, NK-1R, was evaluated using real-time qRT-PCR.

    Results: Our results showed that denervated tendons expressed morphological changes such as hypercellularity; disfigured cells; disorganization of the collagen network; increased production of type III collagen; and increased expression of NK-1R.

    Conclusion: Taken together these data provide new insights into the histopathology of denervated tendons showing that denervation causes somewhat similar changes in the Achilles tendon as does tendinosis in rats.

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  • 26.
    El-Habta, Roine
    et al.
    Umeå University, Faculty of Medicine, Department of Integrative Medical Biology (IMB), Anatomy.
    Kingham, Paul J.
    Umeå University, Faculty of Medicine, Department of Integrative Medical Biology (IMB), Anatomy.
    Backman, Ludvig J.
    Umeå University, Faculty of Medicine, Department of Integrative Medical Biology (IMB), Anatomy.
    Adipose stem cells enhance myoblast proliferation via acetylcholine and extracellular signal-regulated kinase 1/2 signaling2018In: Muscle and Nerve, ISSN 0148-639X, E-ISSN 1097-4598, Vol. 57, no 2, p. 305-311Article in journal (Refereed)
    Abstract [en]

    Introduction: In this study we investigated the interaction between adipose tissue-derived stem cells (ASCs) and myoblasts in co-culture experiments. Methods: Specific inductive media were used to differentiate ASCs in vitro into a Schwann cell-like phenotype (differentiated adipose tissuederived stem cells, or dASCs) and, subsequently, the expression of acetylcholine (ACh)-related machinery was determined. In addition, the expression of muscarinic ACh receptors was examined in denervated rat gastrocnemius muscles. Results: In contrast to undifferentiated ASCs, dASCs expressed more choline acetyltransferase and vesicular acetylcholine transporter. When co-cultured with myoblasts, dASCs enhanced the proliferation rate, as did ACh administration alone. Western blotting and pharmacological inhibitor studies showed that phosphorylated extracellular signal-regulated kinase 1/2 signaling mediated these effects. In addition, denervated muscle showed higher expression of muscarinic ACh receptors than control muscle. Discussion: Our findings suggest that dASCs promote proliferation of myoblasts through paracrine secretion of ACh, which could explain some of their regenerative capacity in vivo.

  • 27.
    El-Habta, Roine
    et al.
    Umeå University, Faculty of Medicine, Department of Integrative Medical Biology (IMB), Anatomy.
    Sloniecka, Marta
    Umeå University, Faculty of Medicine, Department of Integrative Medical Biology (IMB), Anatomy.
    Kingham, Paul J.
    Umeå University, Faculty of Medicine, Department of Integrative Medical Biology (IMB), Anatomy.
    Backman, Ludvig J.
    Umeå University, Faculty of Medicine, Department of Integrative Medical Biology (IMB), Anatomy.
    The adipose tissue stromal vascular fraction secretome enhances the proliferation but inhibits the differentiation of myoblasts2018In: Stem Cell Research & Therapy, E-ISSN 1757-6512, Vol. 9, article id 352Article in journal (Refereed)
    Abstract [en]

    Background: Adipose tissue is an excellent source for isolation of stem cells for treating various clinical conditions including injuries to the neuromuscular system. Many previous studies have focused on differentiating these adipose stem cells (ASCs) towards a Schwann cell-like phenotype (dASCs), which can enhance axon regeneration and reduce muscle atrophy. However, the stromal vascular fraction (SVF), from which the ASCs are derived, also exerts broad regenerative potential and might provide a faster route to clinical translation of the cell therapies for treatment of neuromuscular disorders.

    Methods: The aim of this study was to establish the effects of SVF cells on the proliferation and differentiation of myoblasts using indirect co-culture experiments. A Growth Factor PCR Array was used to compare the secretomes of SVF and dASCs, and the downstream signaling pathways were investigated.

    Results: SVF cells, unlike culture-expanded dASCs, expressed and secreted hepatocyte growth factor (HGF) at concentrations sufficient to enhance the proliferation of myoblasts. Pharmacological inhibitor studies revealed that the signal is mediated via ERK1/2 phosphorylation and that the effect is significantly reduced by the addition of 100 pM Norleual, a specific HGF inhibitor. When myoblasts were differentiated into multinucleated myotubes, the SVF cells reduced the expression levels of fast-type myosin heavy chain (MyHC2) suggesting an inhibition of the differentiation process.

    Conclusions: In summary, this study shows the importance of HGF as a mediator of the SVF effects on myoblasts and provides further evidence for the importance of the secretome in cell therapy and regenerative medicine applications.

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  • 28.
    Fong, Gloria
    et al.
    Umeå University, Faculty of Medicine, Department of Integrative Medical Biology (IMB), Anatomy.
    Backman, Ludvig
    Umeå University, Faculty of Medicine, Department of Integrative Medical Biology (IMB), Anatomy. Umeå University, Faculty of Medicine, Department of Surgical and Perioperative Sciences.
    Andersson, Gustav
    Umeå University, Faculty of Medicine, Department of Integrative Medical Biology (IMB), Anatomy.
    Scott, Alexander
    Vancouver Coastal Health and Research Institute.
    Danielson, Patrik
    Umeå University, Faculty of Medicine, Department of Integrative Medical Biology (IMB), Anatomy.
    Human tenocytes are stimulated to proliferate by acetylcholine through an EGFR signalling pathway2013In: Cell and Tissue Research, ISSN 0302-766X, E-ISSN 1432-0878, Vol. 351, no 3, p. 465-475Article in journal (Refereed)
    Abstract [en]

    Studies of human patellar and Achilles tendons have shown that primary tendon fibroblasts (tenocytes) not only have the capacity to produce acetylcholine (ACh) but also express muscarinic ACh receptors (mAChRs) through which ACh can exert its effects. In patients with tendinopathy (chronic tendon pain) with tendinosis, the tendon tissue is characterised by hypercellularity and angiogenesis, both of which might be influenced by ACh. In this study, we have tested the hypothesis that ACh increases the proliferation rate of tenocytes through mAChR stimulation and have examined whether this mechanism operates via the extracellular activation of the epidermal growth factor receptor (EGFR), as shown in other fibroblastic cells. By use of primary human tendon cell cultures, we identified cells expressing vimentin, tenomodulin and scleraxis and found that these cells also contained enzymes related to ACh synthesis and release (choline acetyltransferase and vesicular acetylcholine transporter). The cells furthermore expressed mAChRs of several subtypes. Exogenously administered ACh stimulated proliferation and increased the viability of tenocytes in vitro. When the cells were exposed to atropine (an mAChR antagonist) or the EGFR inhibitor AG1478, the proliferative effect of ACh decreased. Western blot revealed increased phosphorylation, after ACh stimulation, for both EGFR and the extracellular-signal-regulated kinases 1 and 2. Given that tenocytes have been shown to produce ACh and express mAChRs, this study provides evidence of a possible autocrine loop that might contribute to the hypercellularity seen in tendinosis tendon tissue.

  • 29.
    Fong, Gloria
    et al.
    Umeå University, Faculty of Medicine, Department of Integrative Medical Biology (IMB), Anatomy. Centre for Hip Health and Mobility, Vancouver Coastal Health Research Institute, Vancouver, BC, Canada.
    Backman, Ludvig J.
    Umeå University, Faculty of Medicine, Department of Integrative Medical Biology (IMB), Anatomy.
    Alfredson, Håkan
    Umeå University, Faculty of Medicine, Department of Community Medicine and Rehabilitation, Sports medicine.
    Scott, Alex
    Centre for Hip Health and Mobility, Vancouver Coastal Health Research Institute, Vancouver, BC, Canada.
    Danielson, Patrik
    Umeå University, Faculty of Medicine, Department of Integrative Medical Biology (IMB), Anatomy. Umeå University, Faculty of Medicine, Department of Clinical Sciences, Ophthalmology.
    The Effects of Substance P and Acetylcholine on Human Tenocyte Proliferation Converge Mechanistically via TGF-β12017In: PLOS ONE, E-ISSN 1932-6203, Vol. 12, no 3, article id e0174101Article in journal (Refereed)
    Abstract [en]

    Previous in vitro studies on human tendon cells (tenocytes) have demonstrated that the exogenous administration of substance P (SP) and acetylcholine (ACh) independently result in tenocyte proliferation, which is a prominent feature of tendinosis. Interestingly, the possible link between SP and ACh has not yet been explored in human tenocytes. Recent studies in other cell types demonstrate that both SP and ACh independently upregulate TGF-β1 expression via their respective receptors, the neurokinin 1 receptor (NK-1R) and muscarinic ACh receptors (mAChRs). Furthermore, TGF-β1 has been shown to downregulate NK-1R expression in human keratocytes. The aim of this study was to examine if TGF-β1 is the intermediary player involved in mediating the proliferative pathway shared by SP and ACh in human tenocytes. The results showed that exogenous administration of SP and ACh both caused significant upregulation of TGF-β1 at the mRNA and protein levels. Exposing cells to TGF-β1 resulted in increased cell viability of tenocytes, which was blocked in the presence of the TGFβRI/II kinase inhibitor. In addition, the proliferative effects of SP and ACh on tenocytes were reduced by the TGFβRI/II kinase inhibitor; this supports the hypothesis that the proliferative effects of these signal substances are mediated via the TGF-β axis. Furthermore, exogenous TGF-β1 downregulated NK-1R and mAChRs expression at both the mRNA and protein levels, and these effects were negated by simultaneous exposure to the TGFβRI/II kinase inhibitor, suggesting a negative feedback loop. In conclusion, the results indicate that TGF-β1 is the intermediary player through which the proliferative actions of both SP and ACh converge mechanistically.

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  • 30.
    Fong, Gloria
    et al.
    Umeå University, Faculty of Medicine, Department of Integrative Medical Biology (IMB), Anatomy.
    Backman, Ludvig J.
    Umeå University, Faculty of Medicine, Department of Integrative Medical Biology (IMB), Anatomy.
    Hart, David A.
    Vancouver Coastal Hlth & Res Inst, Ctr Hip Hlth & Mobil, Vancouver, BC, Canada.
    Danielson, Patrik
    Umeå University, Faculty of Medicine, Department of Integrative Medical Biology (IMB), Anatomy.
    McCormack, Bob
    Vancouver Coastal Hlth & Res Inst, Ctr Hip Hlth & Mobil, Vancouver, BC, Canada.
    Scott, Alex
    Univ British Columbia, Dept Phys Therapy, Vancouver, BC V5Z 1M9, Canada.
    Substance P enhances collagen remodeling and MMP-3 expression by human tenocytes2013In: Journal of Orthopaedic Research, ISSN 0736-0266, E-ISSN 1554-527X, Vol. 31, no 1, p. 91-98Article in journal (Refereed)
    Abstract [en]

    The loss of collagen organization is considered a hallmark histopathologic feature of tendinosis. At the cellular level, tenocytes have been shown to produce signal substances that were once thought to be restricted to neurons. One of the main neuropeptides implicated in tendinosis, substance P (SP), is known to influence collagen organization, particularly after injury. The aim of this study was to examine the influence of SP on collagen remodeling by primary human tendon cells cultured in vitro in three-dimensional collagen lattices. We found that SP stimulation led to an increased rate of collagen remodeling mediated via the neurokinin-1 receptor (NK-1 R), the preferred cell receptor for SP. Gene expression analysis showed that SP stimulation resulted in significant increases in MMP3, COL3A1 and ACTA2 mRNA levels in the collagen lattices. Furthermore, cyclic tensile loading of tendon cell cultures along with the administration of exogenous SP had an additive effect on MMP3 expression. Immunoblotting confirmed that SP increased MMP3 protein levels via the NK-1 R. This study indicates that SP, mediated via NK-1 R, increases collagen remodeling and leads to increased MMP3 mRNA and protein expression that is further enhanced by cyclic mechanical loading.

  • 31.
    Jonsson, Frida
    et al.
    Umeå University, Faculty of Medicine, Department of Medical Biosciences, Medical and Clinical Genetics.
    Byström, Berit
    Umeå University, Faculty of Medicine, Department of Clinical Sciences, Ophthalmology.
    Davidson, Alice E.
    UCL Institute of Ophthalmology, London, UK.
    Backman, Ludvig J.
    Umeå University, Faculty of Medicine, Department of Integrative Medical Biology (IMB), Anatomy.
    Kellgren, Therese
    Umeå University, Faculty of Science and Technology, Department of Mathematics and Mathematical Statistics.
    Tuft, Stephen J.
    UCL Institute of Ophthalmology, London, UK; Moorfields Eye Hospital, London, UK.
    Koskela, Timo
    Koskelas Eye Clinic, Umeå, Sweden.
    Ryden, Patrik
    Umeå University, Faculty of Science and Technology, Department of Mathematics and Mathematical Statistics.
    Sandgren, Ola
    Umeå University, Faculty of Medicine, Department of Clinical Sciences, Ophthalmology.
    Danielson, Patrik
    Umeå University, Faculty of Medicine, Department of Integrative Medical Biology (IMB), Anatomy.
    Hardcastle, Alison J.
    UCL Institute of Ophthalmology, London, UK.
    Golovleva, Irina
    Umeå University, Faculty of Medicine, Department of Medical Biosciences, Medical and Clinical Genetics.
    Mutations in Collagen, Type XVII, Alpha 1 (COL17A1) Cause Epithelial Recurrent Erosion Dystrophy (ERED)2015In: Human Mutation, ISSN 1059-7794, E-ISSN 1098-1004, Vol. 36, no 4, p. 463-473Article in journal (Refereed)
    Abstract [en]

    Corneal dystrophies are a clinically and genetically heterogeneous group of inherited disorders that bilaterally affect corneal transparency. They are defined according to the corneal layer affected and by their genetic cause. In this study, we identified a dominantly inherited epithelial recurrent erosion dystrophy (ERED)-like disease that is common in northern Sweden. Whole-exome sequencing resulted in the identification of a novel mutation, c.2816C>T, p.T939I, in the COL17A1 gene, which encodes collagen type XVII alpha 1. The variant segregated with disease in a genealogically expanded pedigree dating back 200 years. We also investigated a unique COL17A1 synonymous variant, c.3156C>T, identified in a previously reported unrelated dominant ERED-like family linked to a locus on chromosome 10q23-q24 encompassing COL17A1. We show that this variant introduces a cryptic donor site resulting in aberrant pre-mRNA splicing and is highly likely to be pathogenic. Bi-allelic COL17A1 mutations have previously been associated with a recessive skin disorder, junctional epidermolysis bullosa, with recurrent corneal erosions being reported in some cases. Our findings implicate presumed gain-of-function COL17A1 mutations causing dominantly inherited ERED and improve understanding of the underlying pathology.

  • 32.
    Li, Junhong
    et al.
    Umeå University, Faculty of Medicine, Department of Integrative Medical Biology (IMB).
    Zhou, Xin
    Umeå University, Faculty of Medicine, Department of Community Medicine and Rehabilitation, Section of Physiotherapy.
    Chen, Jialin
    School of Medicine, Southeast University, Nanjing, China.
    Eliasson, Pernilla
    Department of Orthopaedics, Sahlgrenska University Hospital, Gothenburg, Sweden.
    Kingham, Paul J.
    Umeå University, Faculty of Medicine, Department of Integrative Medical Biology (IMB).
    Backman, Ludvig J.
    Umeå University, Faculty of Medicine, Department of Integrative Medical Biology (IMB). Umeå University, Faculty of Medicine, Department of Community Medicine and Rehabilitation, Section of Physiotherapy.
    Secretome from myoblasts statically loaded at low intensity promotes tenocyte proliferation via the IGF-1 receptor pathway2023In: The FASEB Journal, ISSN 0892-6638, E-ISSN 1530-6860, Vol. 37, no 10, article id e23203Article in journal (Refereed)
    Abstract [en]

    Exercise is widely recognized as beneficial for tendon healing. Recently, it has been described that muscle-derived molecules secreted in response to static exercise influence tendon healing. In this study, the optimal static loading intensity for tendon healing and the composition of secretome released by myoblasts in response to different intensities of static strain were investigated. In an in vitro coculture model, myoblasts were mechanically loaded using a Flexcell Tension System. Tenocytes were seeded on transwell inserts that allowed communication between the tenocytes and myoblasts without direct contact. Proliferation and migration assays, together with RNA sequencing, were used to determine potential cellular signaling pathways. The secretome from myoblasts exposed to 2% static loading increased the proliferation and migration of the cocultured tenocytes. RNA-seq analysis revealed that this loading condition upregulated the expression of numerous genes encoding secretory proteins, including insulin-like growth factor-1 (IGF-1). Confirmation of IGF-1 expression and secretion was carried out using qPCR and enzyme-linked immunosorbt assay (ELISA), revealing a statistically significant upregulation in response to 2% static loading in comparison to both control conditions and higher loading intensities of 5% and 10%. Addition of an inhibitor of the IGF-1 receptor (PQ401) to the tenocytes significantly reduced myoblast secretome-induced tenocyte proliferation. In conclusion, IGF-1 may be an important molecule in the statically loaded myoblast secretome, which is responsible for influencing tenocytes during exercise-induced healing.

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  • 33.
    Mo, Qingyun
    et al.
    School of Medicine, Southeast University, Nanjing, China.
    Zhang, Wei
    School of Medicine, Southeast University, Nanjing, China; Jiangsu Key Laboratory for Biomaterials and Devices, Southeast University, Nanjing, China; China Orthopedic Regenerative Medicine Group (CORMed), Hangzhou, China.
    Zhu, Aijing
    School of Medicine, Southeast University, Nanjing, China.
    Backman, Ludvig J.
    Umeå University, Faculty of Medicine, Department of Integrative Medical Biology (IMB), Anatomy. Umeå University, Faculty of Medicine, Department of Community Medicine and Rehabilitation, Section of Physiotherapy.
    Chen, Jialin
    School of Medicine, Southeast University, Nanjing, China; Jiangsu Key Laboratory for Biomaterials and Devices, Southeast University, Nanjing, China; China Orthopedic Regenerative Medicine Group (CORMed), Hangzhou, China.
    Regulation of osteogenic differentiation by the pro-inflammatory cytokines IL-1β and TNF-α: current conclusions and controversies2022In: Human Cell, ISSN 0914-7470, Vol. 35, p. 957-971Article, review/survey (Refereed)
    Abstract [en]

    Treatment of complex bone fracture diseases is still a complicated problem that is urged to be solved in orthopedics. In bone tissue engineering, the use of mesenchymal stromal/stem cells (MSCs) for tissue repair brings hope to the medical field of bone diseases. MSCs can differentiate into osteoblasts and promote bone regeneration. An increasing number of studies show that the inflammatory microenvironment affects the osteogenic differentiation of MSCs. It is shown that TNF-α and IL-1β play different roles in the osteogenic differentiation of MSCs via different signal pathways. The main factors that affect the role of TNF-α and IL-1β in osteogenic differentiation of MSCs include concentration and the source of stem cells (different species and different tissues). This review in-depth analyzes the roles of pro-inflammatory cytokines in the osteogenic differentiation of MSCs and reveals some current controversies to provide a reference of comprehensively understanding.

  • 34.
    Mousavizadeh, Rouhollah
    et al.
    Department of Medicine, University of British Columbia, Vancouver, BC, Canada.
    Backman, Ludvig
    Umeå University, Faculty of Medicine, Department of Integrative Medical Biology (IMB), Anatomy.
    McCormack, Robert G
    Department of Orthopedic Surgery, University of British Columbia, Vancouver, BC, Canada.
    Scott, Alex
    Vancouver Coastal Health Research Institute, University of British Columbia, Vancouver, BC, Canada.
    Dexamethasone decreases substance P expression in human tendon cells: an in vitro study2015In: Rheumatology, ISSN 1462-0324, E-ISSN 1462-0332, Vol. 54, no 2, p. 318-323Article in journal (Refereed)
    Abstract [en]

    OBJECTIVE: Glucocorticoid injections are used by rheumatologists to treat chronic tendinopathy. Surprisingly, the mechanisms by which corticosteroids induce pain relief in this condition have not been investigated. Previous studies have shown local substance P (SP) levels to be correlated with tendon pain and tissue pathology. The objective of this study was to determine whether SP production in human tenocytes is modulated by exposure to dexamethasone.

    METHODS: Human tendon fibroblasts were cultured in the presence or absence of dexamethasone (1-400 nM), an inhibitor of the glucocorticoid receptor, RU486, recombinant TGF-β (2.5 or 5.0 ng/ml) or an inhibitor of the TGF-β receptor (A83.01), recombinant human IL-1β and IL-6. Expression levels of the genes encoding for SP (TAC1) and its preferred receptor (NK1R), IL-1α, IL-1β and IL-6 were determined with quantitative PCR and protein levels of SP were examined by EIA and western blot.

    RESULTS: Exposure of human tendon cells to dexamethasone resulted in a time-dependent reduction of mRNA for SP in both hamstrings and Achilles tenocytes, whereas NK1R was unaffected. The reduction of SP mRNA was dependent on signalling through the glucocorticoid receptor. SP protein was substantially decreased by dexamethasone. Dexamethasone also prevented induction of SP by IL-1β and by cyclic mechanical loading.

    CONCLUSION: This study demonstrates that dexamethasone treatment of human tendon fibroblasts reduces the expression of SP through a glucocorticoid receptor-dependent pathway. Drugs interfering with SP signalling could be a future target in the treatment of tendinopathy.

  • 35.
    Prittinen, Juha
    et al.
    Umeå University, Faculty of Medicine, Department of Integrative Medical Biology (IMB).
    Zhou, Xin
    Umeå University, Faculty of Medicine, Department of Integrative Medical Biology (IMB).
    Bano, Fouzia
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology. Umeå University, Faculty of Medicine, Wallenberg Centre for Molecular Medicine at Umeå University (WCMM).
    Backman, Ludvig J.
    Umeå University, Faculty of Medicine, Department of Integrative Medical Biology (IMB). Umeå University, Faculty of Medicine, Department of Community Medicine and Rehabilitation.
    Danielson, Patrik
    Umeå University, Faculty of Medicine, Department of Integrative Medical Biology (IMB). Umeå University, Faculty of Medicine, Department of Clinical Sciences, Ophthalmology.
    Microstructured collagen films for 3D corneal stroma modelling2022In: Connective Tissue Research, ISSN 0300-8207, E-ISSN 1607-8438, Vol. 63, no 5, p. 443-452Article in journal (Refereed)
    Abstract [en]

    Purpose/aim: Corneal injury is a major cause of impaired vision around the globe. The fine structure of the corneal stroma plays a pivotal role in the phenotype and behavior of the embedded cells during homeostasis and healing after trauma or infection. In order to study healing processes in the cornea, it is important to create culture systems that functionally mimic the natural environment.

    Materials and methods: Collagen solution was vitrified on top of a grated film to achieve thin collagen films with parallel microgrooves. Keratocytes (corneal stromal cells) were cultured on the films either as a single layer or as stacked layers of films and cells. SEM and F-actin staining were used to analyze the pattern transference onto the collagen and the cell orientation on the films. Cell viability was analyzed with MTS and live/dead staining. Keratocytes, fibroblasts, and myofibroblasts were cultured to study the pattern’s effect on phenotype.

    Results: A microstructured collagen film-based culture system that guides keratocytes (stromal cells) to their native, layerwise perpendicular orientation in 3D and that can support fibroblasts and myofibroblasts was created. The films are thin and transparent enough to observe cells at least three layers deep. The cells maintain viability in 2D and 3D cultures and the films can support fibroblast and myofibroblast phenotypes.

    Conclusions: The films provide an easily reproducible stroma model that maintains high cell viability and improves the preservation of the keratocyte phenotype in keratocytes that are differentiated to fibroblasts.

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  • 36.
    Roux, Sandrine Le
    et al.
    Umeå University, Faculty of Medicine, Department of Integrative Medical Biology (IMB).
    Borbely, Gabor
    Umeå University, Faculty of Medicine, Department of Integrative Medical Biology (IMB).
    Słoniecka, Marta
    Umeå University, Faculty of Medicine, Department of Integrative Medical Biology (IMB). Umeå University, Faculty of Medicine, Department of Clinical Sciences, Ophthalmology.
    Backman, Ludvig J
    Umeå University, Faculty of Medicine, Department of Integrative Medical Biology (IMB).
    Danielson, Patrik
    Umeå University, Faculty of Medicine, Department of Integrative Medical Biology (IMB). Umeå University, Faculty of Medicine, Department of Clinical Sciences, Ophthalmology.
    Transforming Growth Factor Beta 1 Modulates the Functional Expression of the Neurokinin-1 Receptor in Human Keratocytes2016In: Current Eye Research, ISSN 0271-3683, E-ISSN 1460-2202, Vol. 41, no 8, p. 1035-1043Article in journal (Refereed)
    Abstract [en]

    PURPOSE: Transforming growth factor beta 1 (TGF-β1) is a cytokine involved in a variety of processes, such as differentiation of fibroblasts into myofibroblasts. TGF-β1 has also been shown to delay the internalization of the neurokinin-1 receptor (NK-1 R) after its activation by its ligand, the neuropeptide substance P (SP). NK-1 R comprises two naturally occurring variants, a full-length and a truncated form, triggering different cellular responses. SP has been shown to affect important events in the cornea - such as stimulating epithelial cell proliferation - processes that are involved in corneal wound healing and thus in maintaining the transparency of the corneal stroma. An impaired signaling through NK-1 R could thus impact the visual quality. We hypothesize that TGF-β1 modulates the expression pattern of NK-1 R in human corneal stroma cells, keratocytes. The purpose of this study was to test that hypothesis.

    METHODS: Cultures of primary keratocytes were set up with cells derived from healthy human corneas, obtained from donated transplantation graft leftovers, and characterized by immunocytochemistry and Western blot. Immunocytochemistry for TGF-β receptors and NK-1 R was performed. Gene expression was assessed with real-time polymerase chain reaction (qPCR).

    RESULTS: Expression of TGF-β receptors was confirmed in keratocytes in vitro. Treating the cells with TGF-β1 significantly reduced the gene expression of NK-1 R. Furthermore, immunocytochemistry for NK-1 R demonstrated that it is specifically the expression of the full-length isotype of the receptor that is reduced after treatment with TGF-β1, which was also confirmed with qPCR using a specific probe for the full-length receptor.

    CONCLUSIONS: TGF-β1 down-regulates the gene expression of the full-length variant of NK-1 R in human keratocytes, which might impact its signaling pathway and thus explain the known delay in internalization after activation by SP seen with TGF-β1 treatment.

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  • 37. Scott, Alex
    et al.
    Backman, Ludvig J.
    Umeå University, Faculty of Medicine, Department of Integrative Medical Biology (IMB).
    Speed, Cathy
    Tendinopathy: Update on Pathophysiology2015In: Journal of Orthopaedic and Sports Physical Therapy, ISSN 0190-6011, E-ISSN 1938-1344, Vol. 45, no 11, p. 833-841Article in journal (Refereed)
    Abstract [en]

    Tendinopathy has become the accepted term to describe a spectrum of changes that occur in-damaged and/or diseased tendons. Over the past 2 decades, there have been new insights into tendon pathophysiology of relevance to clinicians, including (1) better characterization of the overuse injury process and the resultant structural and functional disruption in chronically painful tendons, (2) improved understanding of the pathomechanics associated with chronic tendon injury, and (3) greater knowledge about the influence of lifestyle factors and drugs on tendon pathology. The implications of these new insights are discussed.

  • 38. Sheng, Renwang
    et al.
    Jiang, Yujie
    Backman, Ludvig J.
    Umeå University, Faculty of Medicine, Department of Integrative Medical Biology (IMB), Anatomy.
    Zhang, Wei
    Chen, Jialin
    The Application of Mechanical Stimulations in Tendon Tissue Engineering2020In: Stem Cells International, ISSN 1687-9678, ISSN 1687-966X, Vol. 2020, article id 8824783Article, review/survey (Refereed)
    Abstract [en]

    Tendon injury is the most common disease in the musculoskeletal system. The current treatment methods have many limitations, such as poor therapeutic effects, functional loss of donor site, and immune rejection. Tendon tissue engineering provides a new treatment strategy for tendon repair and regeneration. In this review, we made a retrospective analysis of applying mechanical stimulation in tendon tissue engineering, and its potential as a direction of development for future clinical treatment strategies. For this purpose, the following topics are discussed; (1) the context of tendon tissue engineering and mechanical stimulation; (2) the applications of various mechanical stimulations in tendon tissue engineering, as well as their inherent mechanisms; (3) the application of magnetic force and the synergy of mechanical and biochemical stimulation. With this, we aim at clarifying some of the main questions that currently exist in the field of tendon tissue engineering and consequently gain new knowledge that may help in the development of future clinical application of tissue engineering in tendon injury.

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  • 39.
    Sheng, Renwang
    et al.
    School of Medicine, Southeast University, Nanjing, China; Center for Stem Cell and Regenerative Medicine, Southeast University, Nanjing, China.
    Liu, Jia
    School of Medicine, Southeast University, Nanjing, China; Center for Stem Cell and Regenerative Medicine, Southeast University, Nanjing, China.
    Zhang, Wei
    School of Medicine, Southeast University, Nanjing, China; Center for Stem Cell and Regenerative Medicine, Southeast University, Nanjing, China; Jiangsu Key Laboratory for Biomaterials and Devices, Southeast University, Nanjing, China; China Orthopedic Regenerative Medicine Group (CORMed), Hangzhou, China.
    Luo, Yifan
    School of Medicine, Southeast University, Nanjing, China; Center for Stem Cell and Regenerative Medicine, Southeast University, Nanjing, China.
    Chen, Zhixuan
    School of Medicine, Southeast University, Nanjing, China; Center for Stem Cell and Regenerative Medicine, Southeast University, Nanjing, China.
    Chi, Jiayu
    School of Medicine, Southeast University, Nanjing, China; Center for Stem Cell and Regenerative Medicine, Southeast University, Nanjing, China.
    Mo, Qingyun
    School of Medicine, Southeast University, Nanjing, China; Center for Stem Cell and Regenerative Medicine, Southeast University, Nanjing, China.
    Wang, Mingyue
    School of Medicine, Southeast University, Nanjing, China; Center for Stem Cell and Regenerative Medicine, Southeast University, Nanjing, China.
    Sun, Yuzhi
    Center for Stem Cell and Regenerative Medicine, Southeast University, Nanjing, China; Department of Orthopaedic Surgery, Institute of Digital Medicine, Nanjing First Hospital, Nanjing Medical University, Nanjing, China.
    Liu, Chuanquan
    School of Medicine, Southeast University, Nanjing, China; Center for Stem Cell and Regenerative Medicine, Southeast University, Nanjing, China.
    Zhang, Yanan
    School of Medicine, Southeast University, Nanjing, China; Center for Stem Cell and Regenerative Medicine, Southeast University, Nanjing, China.
    Zhu, Yue
    School of Medicine, Southeast University, Nanjing, China; Center for Stem Cell and Regenerative Medicine, Southeast University, Nanjing, China.
    Kuang, Baian
    School of Medicine, Southeast University, Nanjing, China; Center for Stem Cell and Regenerative Medicine, Southeast University, Nanjing, China.
    Yan, Chunguang
    School of Medicine, Southeast University, Nanjing, China.
    Liu, Haoyang
    School of Medicine, Southeast University, Nanjing, China; Center for Stem Cell and Regenerative Medicine, Southeast University, Nanjing, China.
    Backman, Ludvig J.
    Umeå University, Faculty of Medicine, Department of Integrative Medical Biology (IMB), Anatomy.
    Chen, Jialin
    School of Medicine, Southeast University, Nanjing, China; Center for Stem Cell and Regenerative Medicine, Southeast University, Nanjing, China; Jiangsu Key Laboratory for Biomaterials and Devices, Southeast University, Nanjing, China; China Orthopedic Regenerative Medicine Group (CORMed), Hangzhou, China.
    Material stiffness in cooperation with macrophage paracrine signals determines the tenogenic differentiation of mesenchymal stem cells2023In: Advanced Science, E-ISSN 2198-3844, Vol. 10, no 17, article id 2206814Article in journal (Refereed)
    Abstract [en]

    Stiffness is an important physical property of biomaterials that determines stem cell fate. Guiding stem cell differentiation via stiffness modulation has been considered in tissue engineering. However, the mechanism by which material stiffness regulates stem cell differentiation into the tendon lineage remains controversial. Increasing evidence demonstrates that immune cells interact with implanted biomaterials and regulate stem cell behaviors via paracrine signaling; however, the role of this mechanism in tendon differentiation is not clear. In this study, polydimethylsiloxane (PDMS) substrates with different stiffnesses are developed, and the tenogenic differentiation of mesenchymal stem cells (MSCs) exposed to different stiffnesses and macrophage paracrine signals is investigated. The results reveal that lower stiffnesses facilitates tenogenic differentiation of MSCs, while macrophage paracrine signals at these stiffnesses suppress the differentiation. When exposed to these two stimuli, MSCs still exhibit enhanced tendon differentiation, which is further elucidated by global proteomic analysis. Following subcutaneous implantation in rats for 2 weeks, soft biomaterial induces only low inflammation and promotes tendon-like tissue formation. In conclusion, the study demonstrates that soft, rather than stiff, material has a greater potential to guide tenogenic differentiation of stem cells, which provides comprehensive evidence for optimized bioactive scaffold design in tendon tissue engineering.

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  • 40.
    Sloniecka, Marta
    et al.
    Umeå University, Faculty of Medicine, Department of Integrative Medical Biology (IMB).
    Backman, Ludvig J.
    Umeå University, Faculty of Medicine, Department of Integrative Medical Biology (IMB).
    Danielson, Patrik
    Umeå University, Faculty of Medicine, Department of Integrative Medical Biology (IMB).
    Acetylcholine induces proliferation of keratocytes through activation of muscarinic receptors2015In: Investigative Ophthalmology and Visual Science, ISSN 0146-0404, E-ISSN 1552-5783, Vol. 56, no 7, article id 723Article in journal (Other academic)
    Abstract [en]

    Purpose: The corneal wound healing response is a complex process involving cytokine-mediated interactions between epithelial cells, keratocytes of the stroma, corneal nerves, tear film, and cells of the immune system. The outcome of the response plays a critical role in the preservation of corneal transparency after injury. The wound healing cascade includes epithelial surface closure, keratocyte apoptosis, proliferation and migration, formation of myofibroblasts, and stromal remodeling.<br /> Acetylcholine (ACh) is regarded as a classical neurotransmitter. However, cells outside of the nervous system have been shown to contain and release ACh. It has been reported that ACh stimulates fibroblast and epithelial cells to proliferate, has an anti-inflammatory effect in macrophages, and upregulates collagen gene expression in fibroblasts. ACh, its muscarinic receptors (mAChRs) and choline acetyltransferase (ChAT; the enzyme responsible for synthesizing ACh) have been shown to be present in corneal epithelium. However, their role in the corneal stroma and corneal injury has not been extensively studied.<br /> We hypothesize that ACh, upon injury, induces corneal stroma cell proliferation, thus promoting the process of wound healing.

    Methods: Primary human corneal stroma cells were derived from healthy corneas obtained from the local cornea bank. Immunocytochemistry was performed to delineate intracellular presence of ChAT and to characterize mAChRs. Crystal violet and MTS assay were used to asses ACh induced cell proliferation. Expression of the proliferation markers PCNA and Ki67 was analyzed by western blot. To determine what type of ACh receptors are involved in ACh induced proliferation, atropine and mecamylamine were used to block muscarininc or nicotinic ACh receptors, respectively.

    Results: Stromal cells expressed ChAT as well as mAChRs of subtypes M1, M3, M4, and M5. Stimulation of stromal cells with ACh led to increased cell viability and metabolic activity. Expression of PCNA and Ki67 was upregulated in ACh treated cells. Furthermore, mAChRs were the receptor group primarily involved in ACh induced proliferation.

    Conclusions: Corneal stroma cells express ChAT and mAChRs. ACh induces stroma cell proliferation through mainly mAChRs, which suggest that ACh may play an important role in corneal wound healing i.e. wound closure and generation on myofibroblasts.

  • 41.
    Sloniecka, Marta
    et al.
    Umeå University, Faculty of Medicine, Department of Integrative Medical Biology (IMB). Umeå University, Faculty of Medicine, Department of Clinical Sciences, Ophthalmology.
    Backman, Ludvig J.
    Umeå University, Faculty of Medicine, Department of Integrative Medical Biology (IMB).
    Danielson, Patrik
    Umeå University, Faculty of Medicine, Department of Integrative Medical Biology (IMB). Umeå University, Faculty of Medicine, Department of Clinical Sciences, Ophthalmology.
    Antiapoptotic Effect of Acetylcholine in Fas-Induced Apoptosis in Human Keratocytes2016In: Investigative Ophthalmology and Visual Science, ISSN 0146-0404, E-ISSN 1552-5783, Vol. 57, no 14, p. 5892-5902Article in journal (Refereed)
    Abstract [en]

    PURPOSE. To investigate the possible antiapoptotic effect of acetylcholine (ACh) in Fas-mediated apoptosis of primary human keratocytes in vitro, and to explore the underlying mechanism. METHODS. Primary human keratocytes were isolated from healthy corneas. Fas ligand (FasL) was used to induce apoptosis in keratocytes. Cell death was assessed by ELISA. Activity of caspase-3, -7, -8, and -9 was measured with luminescent caspase activity assays. Expression of nuclear factor-kappa B (NF-kappa B) gene was assessed with RT-quantitative (q)PCR. Cytochrome c release apoptosis assay kit was used to extract mitochondria and cytosol. Cytochrome c release, cleavage of Bid, and expression of B-cell lymphoma 2 (Bcl-2) were determined by Western blot. RESULTS. Cell death ELISA revealed that ACh is able to reduce Fas-induced apoptosis in keratocytes. Analysis of the activity of effector caspases-3 and -7 showed that ACh, when added to Fas-treated cells, decreases the activation of both these enzymes. The activity of initiator caspases -8 and -9 also decreased when ACh was added to Fas-treated cells. This antiapoptotic effect of ACh was dependent on ACh concentration and activation of muscarinic ACh receptors. Analysis of the antiapoptotic mechanisms triggered by ACh showed that ACh downregulates expression of FasL-induced NF-kappa B RNA expression, upregulates expression of antiapoptotic protein Bcl-2, downregulates expression of proapoptotic protein Bad, reduces cytochrome c release, and prevents proapoptotic Bid protein cleavage. CONCLUSIONS. Acetylcholine has an antiapoptotic effect in a Fas-apoptosis model of human primary keratocytes in vitro. It is therefore possible that ACh may play a role in corneal wound healing, by modulating its initiation phase.

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  • 42.
    Spang, Christoph
    et al.
    Umeå University, Faculty of Medicine, Department of Integrative Medical Biology (IMB), Anatomy.
    Backman, Ludvig J.
    Umeå University, Faculty of Medicine, Department of Integrative Medical Biology (IMB), Anatomy.
    Le Roux, Sandrine
    Umeå University, Faculty of Medicine, Department of Integrative Medical Biology (IMB), Anatomy.
    Chen, Jialin
    Umeå University, Faculty of Medicine, Department of Integrative Medical Biology (IMB), Anatomy.
    Danielson, Patrik
    Umeå University, Faculty of Medicine, Department of Integrative Medical Biology (IMB), Anatomy. Umeå University, Faculty of Medicine, Department of Clinical Sciences, Ophthalmology.
    Glutamate signaling through the NMDA receptor reduces the expression of scleraxis in plantaris tendon derived cells2017In: BMC Musculoskeletal Disorders, E-ISSN 1471-2474, Vol. 18, article id 218Article in journal (Refereed)
    Abstract [en]

    Background: A body of evidence demonstrating changes to the glutaminergic system in tendinopathy has recently emerged. This hypothesis was further tested by studying the effects of glutamate on the tenocyte phenotype, and the impact of loading and exposure to glucocorticoids on the glutamate signaling machinery.

    Methods: Plantaris tendon tissue and cultured plantaris tendon derived cells were immunohisto-/cytochemically stained for glutamate, N-Methyl-D-Aspartate receptor 1 (NMDAR1) and vesicular glutamate transporter 2 (VGluT2). Primary cells were exposed to glutamate or receptor agonist NMDA. Cell death/viability was measured via LDH/MTS assays, and Western blot for cleaved caspase 3 (c-caspase 3) and cleaved poly (ADP-ribose) polymerase (c-PARP). Scleraxis mRNA (Scx)/protein(SCX) were analyzed by qPCR and Western blot, respectively. A FlexCell system was used to apply cyclic strain. The effect of glucocorticoids was studies by adding dexamethasone (Dex). The mRNA of the glutamate synthesizing enzymes Got1 and Gls, and NMDAR1 protein were measured. Levels of free glutamate were determined by a colorimetric assay.

    Results: Immunoreactions for glutamate, VGluT2, and NMDAR1 were found in tenocytes and peritendinous cells in tissue sections and in cultured cells. Cell death was induced by high concentrations of glutamate but not by NMDA. Scleraxis mRNA/protein was down-regulated in response to NMDA/glutamate stimulation. Cyclic strain increased, and Dex decreased, Gls and Got1 mRNA expression. Free glutamate levels were lower after Dex exposure.

    Conclusions: In conclusion, NMDA receptor stimulation leads to a reduction of scleraxis expression that may be involved in a change of phenotype in tendon cells. Glutamate synthesis is increased in tendon cells in response to strain and decreased by glucocorticoid stimulation. This implies that locally produced glutamate could be involved in the tissue changes observed in tendinopathy.

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  • 43.
    Spang, Christoph
    et al.
    Umeå University, Faculty of Medicine, Department of Integrative Medical Biology (IMB), Anatomy. Dr Alfen Orthopedic Spine Center, 97080 Würzburg, Germany.
    Chen, Jialin
    Umeå University, Faculty of Medicine, Department of Integrative Medical Biology (IMB), Anatomy.
    Backman, Ludvig J.
    Umeå University, Faculty of Medicine, Department of Integrative Medical Biology (IMB), Anatomy.
    The tenocyte phenotype of human primary tendon cells in vitro is reduced by glucocorticoids2016In: BMC Musculoskeletal Disorders, E-ISSN 1471-2474, Vol. 17, article id 467Article in journal (Refereed)
    Abstract [en]

    Background: The use of corticosteroids (e.g., dexamethasone) as treatment for tendinopathy has recently been questioned as higher risks for ruptures have been observed clinically. In vitro studies have reported that dexamethasone exposed tendon cells, tenocytes, show reduced cell viability and collagen production. Little is known about the effect of dexamethasone on the characteristics of tenocytes. Furthermore, there are uncertainties about the existence of apoptosis and if the reduction of collagen affects all collagen subtypes.

    Methods: We evaluated these aspects by exposing primary tendon cells to dexamethasone (Dex) in concentrations ranging from 1 to 1000 nM. Gene expression of the specific tenocyte markers scleraxis (Scx) and tenomodulin (Tnmd) and markers for other mesenchymal lineages, such as bone (AlplOcn), cartilage (AcanSox9) and fat (CebpαPparg) was measured via qPCR. Cell viability and proliferation was calculated using a MTS Assay. Cell death was measured by LDH assay and cleaved caspase-3 using Western Blot. Gene expression of collagen subtypes Col1Col3 and Col14 was analyzed using qPCR.

    Results: Stimulation with Dex decreased cell viability and LDH levels. Dex also induced a significant reduction of Scx gene expression and a marked loss of fibroblast like cell shape. The mRNA for all examined collagen subtypes was found to be down-regulated. Among non-tendinous genes only Pparg was significantly increased, whereas AcanAlpl and Sox9 were reduced.

    Conclusions: These results indicate a Dex induced phenotype drift of the tenocytes by reducing scleraxis expression. Reduction of several collagen subtypes, but not cell death, seems to be a feature of Dex induced tissue degeneration.

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  • 44.
    Słoniecka, Marta
    et al.
    Umeå University, Faculty of Medicine, Department of Integrative Medical Biology (IMB).
    Backman, Ludvig J
    Umeå University, Faculty of Medicine, Department of Integrative Medical Biology (IMB).
    Danielson, Patrik
    Umeå University, Faculty of Medicine, Department of Integrative Medical Biology (IMB).
    Acetylcholine enhances keratocyte proliferation through muscarinic receptor activation.2015In: International Immunopharmacology, ISSN 1567-5769, E-ISSN 1878-1705, Vol. 29, no 1, p. 57-62Article in journal (Refereed)
    Abstract [en]

    Acetylcholine (ACh), a classical neurotransmitter, has been shown to be present in various non-neuronal cells, including cells of the eye, such as corneal epithelium and endothelium, and to have widespread physiological effects such as cytoskeleton reorganization, cellular proliferation, differentiation, and apoptosis. The aim of this study was to investigate the effect of ACh on corneal keratocyte proliferation, and the underlying mechanisms, in order to explore its possible effect in corneal wound healing. Primary culture of human keratocytes was established from donated corneas. Cell viability and fraction of proliferating cells were detected by MTS assay and BrdU incorporation ELISA, respectively. Expression of proliferative markers, PCNA and Ki-67, was detected by western blot and immunocytochemistry. Activation of the MAPK/Erk signaling pathway and its involvement in ACh-enhanced proliferation was determined by western blot analysis, MTS, and BrdU ELISA. We found that ACh enhanced keratocyte proliferation even at low concentrations. Stimulation of proliferation was mediated through activation of muscarinic ACh receptors (mAChRs). Western blot analysis revealed that ACh stimulation of keratocytes upregulated the expression of PCNA and Ki-67, and Ki-67 immunocytochemistry showed that ACh-treated cells were in an active phase of the cell cycle. ACh activated MAPK signaling, and this step was crucial for the ACh-enhanced proliferation, as inhibition of the MAPK pathway resulted in ACh having no proliferative effect. In conclusion, ACh enhances keratocyte proliferation and might thus play a role in proper corneal wound healing.

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  • 45. Ward, Ella Rose
    et al.
    Andersson, Gustav
    Umeå University, Faculty of Medicine, Department of Integrative Medical Biology (IMB), Anatomy. Umeå University, Faculty of Medicine, Department of Surgical and Perioperative Sciences, Hand Surgery.
    Backman, Ludvig J.
    Umeå University, Faculty of Medicine, Department of Integrative Medical Biology (IMB), Anatomy.
    Gaida, Jamie E.
    Fat pads adjacent to tendinopathy: more than a coincidence?2016In: British Journal of Sports Medicine, ISSN 0306-3674, E-ISSN 1473-0480, Vol. 50, no 24, p. 1491-1492Article in journal (Refereed)
  • 46.
    Zhang, Aini
    et al.
    School of Medicine, Southeast University, Nanjing, China.
    Zhang, Wei
    School of Medicine, Southeast University, Nanjing, China; Jiangsu Key Laboratory for Biomaterials and Devices Southeast University, Nanjing, China; China Orthopedic Regenerative Medicine Group, China.
    Backman, Ludvig J.
    Umeå University, Faculty of Medicine, Department of Integrative Medical Biology (IMB), Anatomy. Umeå University, Faculty of Medicine, Department of Community Medicine and Rehabilitation, Section of Physiotherapy.
    Chen, Jialin
    School of Medicine, Southeast University, Nanjing, China; Jiangsu Key Laboratory for Biomaterials and Devices Southeast University, Nanjing, China; China Orthopedic Regenerative Medicine Group, China.
    Advances in Regulatory Strategies of Differentiating Stem Cells towards Keratocytes2022In: Stem Cells International, ISSN 1687-9678, Vol. 2022, article id 5403995Article, review/survey (Refereed)
    Abstract [en]

    Corneal injury is a commonly encountered clinical problem which led to vision loss and impairment that affects millions of people worldwide. Currently, the available treatment in clinical practice is corneal transplantation, which is limited by the accessibility of donors. Corneal tissue engineering appears to be a promising alternative for corneal repair. However, current experimental strategies of corneal tissue engineering are insufficient due to inadequate differentiation of stem cell into keratocytes and thus cannot be applied in clinical practice. In this review, we aim to clarify the role and effectiveness of both biochemical factors, physical regulation, and the combination of both to induce stem cells to differentiate into keratocytes. We will also propose novel perspectives of differentiation strategy that may help to improve the efficiency of corneal tissue engineering.

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  • 47.
    Zhang, Wei
    et al.
    Umeå University, Faculty of Medicine, Department of Integrative Medical Biology (IMB), Anatomy.
    Chen, Jialin
    Umeå University, Faculty of Medicine, Department of Integrative Medical Biology (IMB), Anatomy.
    Backman, Ludvig J.
    Umeå University, Faculty of Medicine, Department of Integrative Medical Biology (IMB), Anatomy.
    Malm, Adam D.
    Umeå University, Faculty of Medicine, Department of Integrative Medical Biology (IMB), Anatomy.
    Danielson, Patrik
    Umeå University, Faculty of Medicine, Department of Clinical Sciences, Ophthalmology.
    Surface Topography and Mechanical Strain Promote Keratocyte Phenotype and Extracellular Matrix Formation in a Biomimetic 3D Corneal Model2017In: Advanced Healthcare Materials, ISSN 2192-2640, E-ISSN 2192-2659, Vol. 6, no 5, article id 1601238Article in journal (Refereed)
    Abstract [en]

    The optimal functionality of the native corneal stroma is mainly dependent on the well-ordered arrangement of extracellular matrix (ECM) and the pressurized structure. In order to develop an in vitro corneal model, it is crucial to mimic the in vivo microenvironment of the cornea. In this study, the influence of surface topography and mechanical strain on keratocyte phenotype and ECM formation within a biomimetic 3D corneal model is studied. By modifying the surface topography of materials, it is found that patterned silk fibroin film with 600 grooves mm(-1) optimally supports cell alignment and ECM arrangement. Furthermore, treatment with 3% dome-shaped mechanical strain, which resembles the shape and mechanics of native cornea, significantly enhances the expression of keratocyte markers as compared to flat-shaped strain. Accordingly, a biomimetic 3D corneal model, in the form of a collagen-modified, silk fibroin-patterned construct subjected to 3% dome-shaped strain, is created. Compared to traditional 2D cultures, it supports a significantly higher expression of keratocyte and ECM markers, and in conclusion better maintains keratocyte phenotype, alignment, and fusiform cell shape. Therefore, the novel biomimetic 3D corneal model developed in this study serves as a useful in vitro 3D culture model to improve current 2D cultures for corneal studies.

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  • 48.
    Zhang, Wei
    et al.
    Umeå University, Faculty of Medicine, Department of Integrative Medical Biology (IMB), Anatomy. School of Medicine, Southeast University, Nanjing , China; Jiangsu Key Laboratory for Biomaterials and Devices, Southeast University, Nanjing , China.
    Chen, Jialin
    Umeå University, Faculty of Medicine, Department of Integrative Medical Biology (IMB), Anatomy. School of Medicine, Southeast University, Nanjing , China; Jiangsu Key Laboratory for Biomaterials and Devices, Southeast University, Nanjing , China.
    Qu, Mingli
    Backman, Ludvig J.
    Umeå University, Faculty of Medicine, Department of Integrative Medical Biology (IMB), Anatomy.
    Zhang, Aini
    Liu, Haoyang
    Zhang, Xiaoping
    Zhou, Qingjun
    Danielson, Patrik
    Umeå University, Faculty of Medicine, Department of Integrative Medical Biology (IMB), Anatomy. Umeå University, Faculty of Medicine, Department of Clinical Sciences, Ophthalmology.
    Sustained Release of TPCA-1 from Silk Fibroin Hydrogels Preserves Keratocyte Phenotype and Promotes Corneal Regeneration by Inhibiting Interleukin-1β Signaling2020In: Advanced Healthcare Materials, ISSN 2192-2640, E-ISSN 2192-2659, Vol. 9, no 17, article id 2000591Article in journal (Refereed)
    Abstract [en]

    Corneal injury due to ocular trauma or infection is one of the most challenging vision impairing pathologies that exists. Many studies focus on the pro-inflammatory and pro-angiogenic effects of interleukin-1 beta(IL-1 beta) on corneal wound healing. However, the effect of IL-1 beta on keratocyte phenotype and corneal repair, as well as the underlying mechanisms, is not clear. This study reports, for the first time, that IL-1 beta induces phenotype changes of keratocytes in vitro, by significantly down-regulating the gene and protein expression levels of keratocyte markers (Keratocan, Lumican, Aldh3a1 and CD34). Furthermore, it is found that the NF-kappa B pathway is involved in the IL-1 beta-induced changes of keratocyte phenotype, and that the selective IKK beta inhibitor TPCA-1, which inhibits NF-kappa B, can preserve keratocyte phenotype under IL-1 beta simulated pathological conditions in vitro. By using a murine model of corneal injury, it is shown that sustained release of TPCA-1 from degradable silk fibroin hydrogels accelerates corneal wound healing, improves corneal transparency, enhances the expression of keratocyte markers, and supports the regeneration of well-organized epithelium and stroma. These findings provide insights not only into the pathophysiological mechanisms of corneal wound healing, but also into the potential development of new treatments for patients with corneal injuries.

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  • 49. Zhang, Wei
    et al.
    Ling, Chen
    Liu, Haoyang
    Zhang, Aini
    Mao, Lu
    Wang, Jing
    Chao, Jie
    Backman, Ludvig J.
    Umeå University, Faculty of Medicine, Department of Integrative Medical Biology (IMB), Anatomy.
    Yao, Qingqiang
    Chen, Jialin
    Tannic acid-mediated dual peptide-functionalized scaffolds to direct stem cell behavior and osteochondral regeneration2020In: Chemical Engineering Journal, ISSN 1385-8947, E-ISSN 1873-3212, Vol. 396, article id 125232Article in journal (Refereed)
    Abstract [en]

    The development of cell-instructive scaffolds, which provide biochemical cues to direct endogenous bone marrow-derived mesenchymal stem cells (BMSCs) behavior, has the potential to revolutionize osteochondral tissue engineering. However, scaffold material itself is generally lacking the inductive signals. Here, a novel peptide-functionalized scaffold was prepared by prime-coating Ca-alginate scaffold with tannic acid (TA) followed by conjugation of E7/P15 peptides (CA-TA-E7/P15). The system leveraged TA as a reactive intermediate between Ca-alginate and peptides due to the multiple functional groups of TA. These interactions induced by TA prime-coating contributed to enhanced scaffold stability and mechanical properties, increased peptide conjugation and sustained release of peptides without affecting their bioactivity, in a TA concentration-dependent manner. The conjugation of E7/P15 peptides endowed the scaffold with the potential to enhance BMSCs recruitment and deposition of cartilage and bone extracellular matrix (ECM). Furthermore, the prepared CA-TA-E7/P15 scaffold showed a promoted biological performance of simultaneous cartilage and subchondral bone regeneration in rabbit osteochondral defect model. These findings indicate that TA is an effective surface modification intermediate and crosslinking aid, and that the CA-TA-E7/P15 scaffold developed in this study serves as a promising cell-instructive scaffold for osteochondral regeneration.

  • 50.
    Zhou, Xin
    et al.
    Umeå University, Faculty of Medicine, Department of Integrative Medical Biology (IMB).
    Backman, Ludvig J.
    Umeå University, Faculty of Medicine, Department of Integrative Medical Biology (IMB). Umeå University, Faculty of Medicine, Department of Community Medicine and Rehabilitation, Section of Physiotherapy.
    Danielson, Patrik
    Umeå University, Faculty of Medicine, Department of Integrative Medical Biology (IMB). Umeå University, Faculty of Medicine, Department of Clinical Sciences, Ophthalmology.
    Activation of NF-κB signaling via cytosolic mitochondrial RNA sensing in kerotocytes with mitochondrial DNA common deletion2021In: Scientific Reports, E-ISSN 2045-2322, Vol. 11, no 1, article id 7360Article in journal (Refereed)
    Abstract [en]

    Scar formation as a result of corneal wound healing is a leading cause of blindness. It is a challenge to understand why scar formation is more likely to occur in the central part of the cornea as compared to the peripheral part. The purpose of this study was to unravel the underlying mechanisms. We applied RNA-seq to uncover the differences of expression profile in keratocytes in the central/peripheral part of the cornea. The relative quantity of mitochondrial RNA was measured by multiplex qPCR. The characterization of mitochondrial RNA in the cytoplasm was confirmed by immunofluoresence microscope and biochemical approach. Gene expression was analyzed by western blot and RT qPCR. We demonstrate that the occurrence of mitochondrial DNA common deletion is greater in keratocytes from the central cornea as compared to those of the peripheral part. The keratocytes with CD have elevated oxidative stress levels, which leads to the leakage of mitochondrial double-stranded RNA into the cytoplasm. The cytoplasmic mitochondrial double-stranded RNA is sensed by MDA5, which induces NF-κB activation. The NF-κB activation thereafter induces fibrosis-like extracellular matrix expressions and IL-8 mRNA transcription. These results provide a novel explanation of the different clinical outcome in different regions of the cornea during wound healing.

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