Proliferation and differentiation of human tenocytes in response to platelet rich plasma: an in vitro and in vivo study

Platelet rich plasma (PRP) is the autologous plasma fraction with a platelet-rich cellular component which is enriched with a number of growth factors. Due to its availability and low cost, PRP has become an increasingly popular clinical tool as an alternative source of growth factors for various applications, for example, tendon regeneration but with limited success in clinical trials. The main objective of the current study was to determine whether activated PRP [i.e., platelet rich plasma-clot release (PRCR)] could be used to induce the proliferation and collagen synthesis in human tenocyte in vitro. The advantage of using PRCR is that the platelet-derived bioactive factors are more concentrated and could initiate a more rapid and accelerated healing response than PRP. Our results demonstrated that 10% PRCR treatment accelerated the extent of cell proliferation and collagen production by human tenocytes in vitro. The expression of specific tenocyte markers were similar to conventional fetal bovine serum (FBS)-treated tenocytes implanted in mice within 14 days of implantation in diffusion chambers. Moreover, relatively more collagen fibrils were evident in PRCR-treated tenocytes in vivo as compared to 10% FBS-treated cells. Overall, our feasibility study has indicated that PRCR can induce human tenocyte proliferation and collagen synthesis which could be implemented for future tendon regeneration in reconstructive surgeries.     

Extracellular matrix expression of human tenocytes in three‐dimensional air–liquid and PLGA cultures compared with tendon tissue: Implications for tendon tissue engineering

Tenocyte transplantation may prove to be an approach to support healing of tendon defects. Cell–cell and cell–matrix contacts within three‐dimensional (3D) cultures may prevent tenocyte dedifferentiation observed in monolayer (2D) culture. The present study compares both neotissue formation and tenocyte extracellular matrix (ECM) expression in 2D and 3D cultures directly with that of native tendon, in order to determine optimal conditions for tendon tissue engineering. Primary human tenocytes were embedded in poly[lactic‐co‐glycolic‐acid] (PLGA)‐scaffolds and high‐density cultures. Neotissue formation was examined by hematoxyline–eosine (H&E) and immunofluorescence staining. Gene expression of ECM proteins and vascular endothelial growth factor (VEGF) was compared at days 0 (2D), 14, and 28 in 3D cultures and tendon. Histomorphology of 3D culture showed tendon‐like tissue as tenocyte cell nuclei became more elongated and ECM accumulated. Type I collagen gene expression was higher in 2D culture than in tendon and decreased in 4‐week‐old 3D cultures, whereas type III collagen was only elevated in high‐density culture compared with tendon. Decorin and COMP were reduced in 2D and increased in 3D culture almost to ex vivo level. These results suggest that the 3D high‐density or biodegradable scaffolds cultures encourage the differentiation of expanded monolayer tenocytes in vitro to tendon‐like tissue. © 2010 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 28:1170–1177, 2010     

腱细胞与人工材料体外联合培养的形态学观察

目的：为了探索肌腱细胞与人工材料是否有可能复合形成新型有活性的人工肌腱，特设计了本项研究。方法：取第４代腱细胞，复苏后分别加入碳纤维编织带、涤纶编织带及几丁质编织带联合培养，在倒置显微镜及透射电镜下观察形态学改变及细胞与材料的相容性。结果：腱细胞与碳纤维有良好的相容性。腱细胞沿碳纤维生长、繁殖，并合成胶原。腱细胞与涤纶的相容性较差，在几丁质上无细胞附着。结论：腱细胞与碳纤维联合培养后，保持了腱细胞的形态特征，与碳纤维的相容性最好。有可能成为一种新型有活性的人工肌腱材料     

Triamcinolone suppresses human tenocyte cellular activity and collagen synthesis

Glucocorticoid injection is widely used in tendon disorders. Despite previous studies on the histologic and biomechanical changes in tendons after glucocorticoid injections, the role of glucocorticoid in tendon rupture still is controversial. It was hypothesized that glucocorticoid has a direct deleterious effect on human tenocytes, suppressing its cellular activity and collagen production. Primary cultures of human tenocytes were obtained from explants of healthy patellar tendon harvested during anterior cruciate ligament reconstructions. The effects on cell viability and cell proliferation were measured by [3-(4,5-demethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay and 5-bromo-deoxyuridine incorporations. The effect on collagen synthesis was measured by H-proline incorporation assay. Triamcinolone acetonide at 10 to 10 mol/L decreased human tenocyte viability to 45% to 88% of control in a dose-dependent manner. Cell proliferation was suppressed to 87% +/- 8% at all doses. Treatment with 1 micromol/L triamcinolone acetonide reduced the amount of collagen synthesis as measured by H-proline incorporation from 40 +/- 2 cpm/1000 cells to 27 +/- 4 cpm/1000 cells. The suppressed human tenocyte cellular activity and reduced collagen production may lead to disturbed tendon structure and predispose the tendon to subsequent spontaneous rupture.     

Extracorporeal shock waves promote healing of collagenase-induced Achilles tendinitis and increase TGF-beta1 and IGF-I expression.

Extracorporeal shock waves (ESW) have recently been used in resolving tendinitis. However, mechanisms by which ESW promote tendon repair is not fully understood. In this study, we reported that an optimal ESW treatment promoted healing of Achilles tendintis by inducing TGF-beta1 and IGF-I. Rats with the collagenease-induced Achilles tendinitis were given a single ESW treatment (0.16 mJ/mm(2) energy flux density) with 0, 200, 500 and 1000 impulses. Achilles tendons were subjected to biomechanical (load to failure and stiffness), biochemical properties (DNA, glycosaminoglycan and hydroxyproline content) and histological assessment. ESW with 200 impulses restored biomechanical and biochemical characteristics of healing tendons 12 weeks after treatment. However, ESW treatments with 500 and 1000 impulses elicited inhibitory effects on tendinitis repair. Histological observation demonstrated that ESW treatment resolved edema, swelling, and inflammatory cell infiltration in injured tendons. Lesion site underwent intensive tenocyte proliferation, neovascularization and progressive tendon tissue regeneration. Tenocytes at the hypertrophied cellular tissue and newly developed tendon tissue expressed strong proliferating cell nuclear antigen (PCNA) after ESW treatment, suggesting that physical ESW could increase the mitogenic responses of tendons. Moreover, the proliferation of tenocytes adjunct to hypertrophied cell aggregate and newly formed tendon tissue coincided with intensive TGF-beta1 and IGF-I expression. Increasing TGF-beta1 expression was noted in the early stage of tendon repair, and elevated IGF-I expression was persisted throughout the healing period. Together, low-energy shock wave effectively promoted tendon healing. TGF-beta1 and IGF-I played important roles in mediating ESW-stimulated cell proliferation and tissue regeneration of tendon.     

Glucocorticoids inhibit tenocyte proliferation and Tendon progenitor cell recruitment.

Corticosteroid injection is commonly used to treat tendon injuries but is often associated with tendon rupture and impaired tendon healing. The effects of dexamethasone on tenocytes have been studied in vitro but only using high concentrations of dexamethasone in monolayer cultures of tenocytes over short periods of time. We have therefore investigated the effects of physiological and pharmacological concentrations of dexamethasone on monolayer cultures of tenocytes over extended time periods. We have also used fibroblastic-colony forming unit cultures to examine the effects of dexamethasone on a progenitor cell population located in tendons. Culturing tenocytes in the presence of dexamethasone for a period of 24 days resulted in a concentration-related decrease in cell number and collagen synthesis as compared to control cultures. This effect was time dependent with cell number in both dexamethasone-treated and control cultures leveling off after 14 days with the control cultures reaching higher cell densities. In contrast in control cultures, collagen accumulation continued to increase until week 4, whereas in the presence of dexamethasone, this tended to level off after 14 days. To study the role of progenitor cell recruitment, the effects of dexamethasone were investigated using the fibroblastic-colony forming unit assay. Treatment with dexamethasone at concentrations of 0.1 nM to 10 microM leads to a progressive reduction in mean colony size as compared to control cultures. Colony number remained constant at concentrations below 10 nM but fell progressively at concentrations above this. In conclusion, dexamethasone reduces both cell number and collagen synthesis in tenocyte cultures in a concentration-dependent manner by both direct effects on tenocyte proliferation and collagen accumulation, and also by modulating the recruitment of tendon progenitor cells.     

Is cultured tendon fibroblast a good model to study tendon healing?

Cultured tendon fibroblasts (CTFs) from intact explants are widely used to study tendon healing in vitro. The significance of these findings may rely on similarities between CTFs and healing tendon fibroblasts in situ. Our purpose was to compare CTFs with fibroblasts cultured from healing tendons. We cultured CTFs from intact and healing tendons at day 7 and day 14 postinjury in a rat model of patellar donor site injury. The mRNA expression of COL1A1, COL3A1, decorin, and biglycan, with or without supplementation of 1 ng/mL TGF‐β1, was compared by quantitative real‐time RT‐PCR. The expression of proliferation cell nuclear antigen (PCNA) and α‐smooth muscle actin (α‐SMA) was determined by immunostain. COL3A1 and decorin mRNA in CTFs was lower as compared to day 7 healing fibroblasts, but its biglycan mRNA level was higher than day 14 healing fibroblasts. TGF‐β1 increased COL1A1 and decorin mRNA in CTFs, but decreased the mRNA of all four genes in day 7 healing tendon fibroblasts. CTFs exhibited lower PCNA immunopositivity as compared to day 7 and day 14 healing fibroblasts, but a higher α‐SMA immunopositivity than cultured day 14 healing fibroblasts. These findings showed that CTFs did not resemble healing tendon cells with respect to major cellular activities related to tendon healing. Thus, fibroblasts from healing tendon may be a more appropriate model for studying cellular activities in tendon healing. © 2007 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 26:374–383, 2008     

Mechanism of mast cell adhesion to human tenocytes in vitro

Mast cells and fibroblasts are two key players involved in many fibrotic and degenerative disorders. In the present study we examined the nature of binding interactions between human mast cells and tendon fibroblasts (tenocytes). In the mast cell‐fibroblast co‐culture model, mast cells were shown to spontaneously bind to tenocytes, in a process that was partially mediated by α5β1 integrin receptors. The same receptors on mast cells significantly mediated binding of these cells to tissue culture plates in the presence of tenocyte‐conditioned media; the tenocyte‐derived fibronectin in the media was shown to also play a major role in these binding activities. Upon binding to tenocytes or tissue culture plates, mast cells acquired an elongated phenotype, which was dependent on α5β1 integrin and tenocyte fibronectin. Additionally, tenocyte‐derived fibronectin significantly enhanced mRNA expression of the adhesion molecule, THY1, by mast cells. Our data suggests that α5β1 integrin mediates binding of mast cells to human tenocyte and to tenocyte‐derived ECM proteins, in particular fibronectin. © 2014 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 33:9–16, 2015.

     

Low-level laser irradiation stimulates tenocyte proliferation in association with increased NO synthesis and upregulation of PCNA and cyclins

Low-level laser therapy is commonly used to treat tendinopathy or tendon injury. Tendon healing requires tenocyte migration to the repair site, followed by proliferation and synthesis of the extracellular matrix. There are few evidence to elucidate that low-level laser promote tenocyte proliferation. This study was designed to determine the effect of laser on tenocyte proliferation. Furthermore, the association of this effect with secretion of nitric oxide (NO) and the expressions of proliferating cell nuclear antigen (PCNA) and cyclins D1, E, A, and B1 was investigated. Tenocytes intrinsic to rat Achilles tendon were treated with low-level laser (660 nm). Tenocyte proliferation was evaluated by MTT assay and immunocytochemistry with Ki-67 stain. NO in the conditioned medium was measured by ELISA. Western blot analysis was used to evaluate the protein expressions of PCNA and cyclins D1, E, A, and B1. The results revealed that tenocytes proliferation was enhanced dose dependently by laser. NO secretion was increased after laser treatment. PCNA and cyclins E, A, and B1 were upregulated by laser. In conclusion, low-level laser irradiation stimulates tenocyte proliferation in a process that is mediated by upregulation of NO, PCNA, and cyclins E, A, and B1.     

In vitro studies to evaluate the effect of varying culture conditions and IPL fluencies on tenocyte activities

Tendons are dense, fibrous connective tissues which carry out the essential physiological role of transmitting mechanical forces from skeletal muscle to bone. From a clinical perspective, tendinopathy is very common, both within the sporting arena and amongst the sedentary population. Studies have shown that light therapy may stimulate tendon healing, and more recently, intense pulsed light (IPL) has attracted attention as a potential treatment modality for tendinopathy; however, its mechanism of action and effect on the tendon cells (tenocytes) is poorly understood. The present study therefore investigates the influence of IPL on an in vitro bovine tendon model. Tenocytes were irradiated with IPL at different devise settings and under variable culture conditions (e.g. utilising cell culture media with or without the pH indicator dye phenol red), and changes in tenocyte viability and migration were subsequently investigated using Alamar blue and scratch assays, respectively. Our data demonstrated that IPL fluencies of up to 15.9 J/cm² proved harmless to the tenocyte cultures (this was the case using culture media with or without phenol red) and resulted in a significant increase in cell viability under certain culture conditions. Furthermore, IPL treatment of tenocytes did not affect the rate of cell migration. This study demonstrates that irradiation with IPL is not detrimental to the tenocytes and may increase their viability under certain conditions, thus validating our in vitro model. Further studies are required to elucidate the effects of IPL application in the clinical situation.     

Role of pulsed electromagnetic fields (PEMF) on tenocytes and myoblasts—potential application for treating rotator cuff tears

The post‐surgery integrity of the tendons and muscle quality are the two major factors in success of rotator cuff (RC) repair. Though surgical techniques for rotator cuff repair have significantly improved in the past two decades, there are no effective treatments to improve tendon‐to‐bone healing and muscle quality after repair at this point in time. Pulsed electromagnetic fields (PEMF) have previously been used for promoting fracture healing. Previous studies have shown that PEMF has a positive role in promoting osteoblast precursors proliferation and differentiation. However, PEMFs effect on tenocytes and muscle cells has not been determined fully yet. The purpose of this study is to define the role of a commercially available PEMF on tenocytes and myoblasts growth and differentiation in vitro. Human rotator cuff tenocytes and C2C12 murine myoblasts were cultured and treated with PEMF for 2 weeks under regular and inflammatory conditions. Our results showed that 2 weeks treatment of PEMF enhanced gene expressions of growth factors in human rotator cuff tenocytes under inflammatory conditions. PEMF significantly enhanced C2C12 myotube formation under normal and inflammatory conditions. Results from this study suggest that PEMF has a positive role in promoting tenocyte gene expression and myoblast differentiation. Therefore, PEMF may potentially serve as a non‐operative treatment to improve clinical incomes rotator cuff tendon repairs. Results © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 35:956–964, 2017.

     

生物衍生材料构建组织工程肌腱体内植入的实验研究

目的 探讨用同种异体肌腱细胞与生物衍生肌腱材料体外联合培养后植入体内，构建组织工程肌腱的可行性。方法 选用四川锦猴15只，手术造成纤维鞘管内屈指深肌腱2．5cm缺损后，分三组。A组：生物衍生肌腱材料构建的组织工程肌腱移植组；B组：单纯生物衍生肌腱材料移植组；C组：自体肌腱移植组。术后1、2、3、6和12周分期观察植入物的大体形态、组织学和超微结构，BrdU标记表达。结果 A组植入体内后肌腱细胞能继续增殖，细胞形态随着时间增加而逐渐趋于正常，形成的肌腱呈白色且有光泽、致密，组织学可见胶原纤维排列较为规则，12周肌腱细胞仍成活并分泌胶原，BrdU表达为阳性；B组植入体内3周后材料逐渐变细，12周后材料被逐渐降解吸收出现中断；C组植入体内2周后桥接部有纤维连接，排列较为规则，肌腱愈合。A组分别于3、6、12周进行扫描电镜观察可见肌腱细胞排列均匀，胶原纤维相互连接形成网状，主体趋势与肌腱走行方向一致；透射电镜下可见细胞核仁清晰，细胞器丰富。随时间的增加A、C组与B组的差异明显增大。结论 同种异体肌腱细胞与生物衍生肌腱材料复合构建的组织工程肌腱，植入免疫功能正常的动物体内能够再生出肌腱样组织，植入的肌腱细胞具有生命力，新生的肌腱组织在大体、组织学方面与正常肌腱相似。     

MGF enhances tenocyte invasion through MMP‐2 activity via the FAK‐ERK1/2 pathway

Tendon regeneration and healing requires tenocytes to move to the repair site followed by proliferation and synthesis of the extracellular matrix. A novel synthetic growth factor, mechano‐growth factor (MGF), has been discovered to have positive roles in tissue repair through the improvement of cell proliferation and migration and the protection of cells against injury‐induced apoptosis. However, it remains unclear whether MGF has the potential to accelerate tendon repair. In this study, using a transwell system, we found that MGF‐C25E (a synthetic mechano‐growth factor E peptide) significantly promotes tenocyte invasion, which was accompanied by the increased phosphorylation of focal adhesion kinase (FAK) and extracellular signal regulated kinase1/2 (ERK1/2) as well as the increased activity of matrix metalloproteinases‐2 (MMP‐2). The MMP‐2 inhibitor OA‐Hy blocked MGF‐C25E‐promoted tenocyte invasion. Inhibitors of FAK or ERK1/2 blocked MGF‐C25E‐promoted tenocyte invasion and MMP‐2 activity as well. These results indicate that MGF‐C25E promotes tenocyte invasion by increasing MMP‐2 activity via the FAK‐ERK1/2 signaling pathway. Taken together, our findings provide the first evidence that MGF‐C25E enhances tenocyte invasion and indicate that it may serve as a potential repair material for promoting the healing and regeneration of injured tendons.     

屈肌腱愈合过程中产生的相对分子质量为45 000的蛋白对腱细胞生长的作用

目的 了解肌腱愈合过程中产生的相对分子质量为45000的蛋白对腱细胞生长、增殖的作用。方法 按Handerson分步酶消化法分离刚孵化的Leghorn鸡的践屈肌腱腱细胞。选用F－12培养基，加入不同浓度的肌腱愈合过程中产生的相对分子质量为45000的蛋白，与腱细胞进行原代培养。通过绘制细胞生长曲线及MTT比色试验，观察该蛋白对腱细胞的作用。结果 屈肌腱愈合过程中产生的相对分子质量为45000的蛋白，可促进腱细胞的增殖，其增殖作用与该蛋白的浓度在一定范围内呈量效关系。结论 屈肌腱愈合过程中产生的相对分子质量为45000的蛋白，对腱细胞的增殖有显著的促进作用。     

Biologic behavior of an in vitro hydrated collagen gel-human tenocyte tendon model

An in vitro human tenocyte-collagen gel model was developed to study tenocyte-mediated Type I collagen fibril reorganization, proliferation, and Type I collagen gene expression. Human tenocytes, obtained from extrasynovial forearm flexor tendons from children 5 to 10 years of age were cultured on plastic or in a cylinder of hydrated Type I collagen gel. Collagen solution was seeded with human tenocytes at 5 x 10(5) cells/mL and gelled in cylinder molds; gel cylinders without human tenocytes served as controls. Gel cylinders were pinned to troughs to create noncompliance. The gel cylinders were analyzed for collagen birefringence and cell shape at 7 and 21 days and for proliferation and gene expression for Type I collagen at 7 days. Under conditions of noncompliance, human tenocytes reoriented Type I collagen into longitudinal bundles resembling the parallel organization of collagen in native tendons. Tenocyte shape became fusiform between the collagen bundles which mimics the morphologic features of a tenocyte in vivo. The structural changes in the tenocytes and matrix are accompanied by downregulation of human tenocyte proliferation and Type I collagen gene expression. When released from the gel cylinder and grown again on plastic, human tenocytes resume proliferation and Type I collagen gene expression. The human tenocytes in this in vitro gel cylinder model system control fibril reorganization and proliferation, resembling their behavior during the development and repair of native tendons.     

Primary cilia are highly oriented with respect to collagen direction and long axis of extensor tendon

Skeletal tissues adapt to their mechanical environments by modulating gene expression, cell metabolism, and extracellular matrix (ECM) architecture; however, the mechanosensory mechanisms for these processes are incompletely understood. Primary cilia have emerged as critical components of the cellular mechanosensory apparatus and have been hypothesized to participate in establishment of cellular and ECM orientation, but their function in skeletal tissues is just beginning to be examined. Here we focused on tendon, a tissue with an oriented matrix that is ideal for analysis of spatial relationships between primary cilia and the ECM. The objective of this study was to characterize the incidence and orientation of tenocyte primary cilia in their native ECM. Primary cilia, nuclei, and collagen were analyzed three‐dimensionally in immunofluorescently labeled rat extensor tendon using multiphoton microscopy and semiautomated morphometry. Primary cilia were observed in 64% of tenocytes. The cilia were highly oriented with respect to the ECM: cilia were aligned parallel to the collagen fibers and the long axis of the tendon. This study represents the first quantification of the in situ incidence and orientation of primary cilia in tendon. © 2009 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 28:77–82, 2010     

Inhibition effect of mannose-6-phosphate on expression of transforming growth factor Beta receptor in flexor tendon cells

Transforming growth factor beta (TGF-β) has an important role in tendon healing and adhesion formation. Inhibiting TGF-β and its receptor expression may prevent adhesions after tendon open. The goal of this study was to examine the effects of mannose-6-phosphate, a natural inhibitor of TGF-β, on TGF-β and its receptor production in tendon sheath fibroblasts, epitenon tenocytes, and endotenon tenocytes of rabbit flexor tendons. Tendon sheath fibroblasts, epitenon tenocytes, and endotenon tenocytes were isolated from rabbit flexor tendons and cultured separately. The cells were divided into 2 groups at random: an experiment group supplemented with mannose-6-phosphate and a control group without mannose-6-phosphate. The expression of TGF-β and TGF-β receptor was quantified with enzyme-linked immunosorbent assay. The luciferase assay measured TGF-β bioactivity. Transforming growth factor beta expression in the experimental group was not decreased compared with the control group, with no significant difference (P>.05) Transforming growth factor beta receptor expression in the experiment group was significantly lower than that in control group (P<.05). Mannose-6-phosphate significantly decreased the expression of TGF-β receptor and TGF-β bioactivity. Modulation of mannose-6-phosphate levels may provide a means of modulating the effects of TGF-β on adhesion formation in flexor tendon wound healing.     

Mechanical stretch promotes tenocyte migration via chromatin remodelling-mediated nuclear morphology changes

Wound healing and function recovery of injured tendons are still a big challenge for orthopaedic surgery. Evidence in clinic shows that early controlled motion has significant favourable effects on tendon healing; however, the mechanisms involved in are not fully understood. In the present study, it was shown that an appropriate mechanical stretch (10% strain, 0.5 Hz for 1 h) evidently promotes rat tenocyte migration and nuclear morphology changes. The farther research discovered that mechanical stretch had no effect on Lamin A/C expression, but it could promote chromatin decondensation. Moreover, the histone modification plays an important role in mechanical stretch-mediated chromatin decondensation. Inhibition histone modification could inhibit mechanical stretch-promoted nuclear morphology changes and tenocyte migration. These results indicating that mechanical stretch may promote tenocyte migration via chromatin remodelling-mediated nuclear morphology changes, which contribute to a better understanding of the role of mechanical stretch on tenocyte migration and repair of injured tendon.     

Effects of Substrate Stiffness on Morphology and MMP-1 Gene Expression in Tenocytes Stimulated With Interleukin-1β

Tendon cells, tenocytes, are constantly subjected to mechanical stress in vivo, which maintains a level of cellular tension. When a tendon is subjected to overloading, local rupture of collagen fibers are induced, which deprives tenocytes of mechanical stress, lowers their cellular tension level and upregulates their catabolism. In addition, leukocytes are attracted to the rupture sites and produce interleukin-1β (IL-1β), and this exogenous IL-1β also stimulates tenocyte catabolism. We tested a hypothesis that catabolic tenocytes with low cellular tension at the rupture sites excessively respond to the exogenous IL-1β and further upregulate matrix metalloproteinase 1 (MMP-1) gene expression. Tenocytes from rabbit Achilles tendon were cultured on the following substrates: glass or polydimethylsiloxane micropillar substrates with a height of 2, 4, or 8 µm. Following a 3-day IL-1β stimulation at a concentration of 0, 1, 10, or 100 pM, the effects of IL-1β stimulation on cell morphology and MMP-1 gene expression was analysed with fluorescent microscopy and fluorescence in situ hybridization, respectively. In addition, the effects of IL-1β stimulation on cell membrane fluidity were examined. It was demonstrated that the cells on 8-µm-height micropillars exhibited a greater response than those on rigid substrates with flat (glass) and topologically the same surface (2-µm-height micropillars) to IL-1β when supplied at the same concentration. Besides this, membrane fluidity was lower in the cells on micropillars. Therefore, it appears that cellular attachment to softer substrates lowers the cellular actin cortex tension, reducing the membrane fluidity and possibly elevating the sensitivity of IL-1 receptors to ligand binding. © 2019 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 38:150–159, 2020