[Piezo1 Mediates the Regulation of Substrate Stiffness on Primary Cilia in Chondrocytes]. / Piezo1介导基底硬度调控软骨细胞初级纤毛形态.

Objective: To investigate how substrate stiffness regulates the morphology of primary cilia in chondrocytes and to illustrate how Piezo1 mediates the morphology regulation of primary cilia by substrate stiffness. Methods: Polydimethylsiloxane (PDMS) curing agent and the main agent (Dow Corning, Beijing, China) were mixed at the ratio of 1∶10 (stiff), 1∶50 (medium stiffness), and 1∶70 (soft), respectively, to prepare substrate films with the thickness of 1 mm at different levels of stiffness, including stiff substrate of (2.21±0.12) MPa, medium-stiffness substrate of (54.47±6.06) kPa, and soft substrate of (2.13±0.10) kPa. Chondrocytes were cultured with the substrates of three different levels of stiffness. Then, the cells were treated with Tubastatin A (Tub A) to inhibit histone deacetylase 6 (HDAC6), Piezo1 activator Yoda1, and inhibitor GsMTx4, respectively. The effects of HDAC6, Yoda1, and GsMTx4 on chondrocyte morphology and the length of primary cilia were analyzed through immunofluorescence staining. Results: The stiff substrate increased the spread area of the chondrocytes. Immunofluorescence assays showed that the cytoskeleton and the nuclear area of the cells on the stiff substrate were significantly increased (P<0.05) and the primary cilia were significantly extended (P<0.05) compared with those on the medium-stiffness and soft substrates. However, the presence rate of primary cilia was not affected. The HDAC6 activity of chondrocytes increased with the decrease in substrate stiffness. When the activity of HDAC6 was inhibited, the cytoskeletal area, the nuclei area, and the primary cilium length were increased more significantly on the stiff substrate (P<0.05). Further testing showed that Piezo1 activator and inhibitor could regulate the activity of HDAC6 in chondrocytes, and that the length of primary cilia was significantly increased after treatment with the activator Yoda1 (P<0.05). On the other hand, the length of primary cilia was significantly shortened on the stiff substrate after treatment with the inhibitor GsMTx4 (P<0.05). Conclusion: Both substrate stiffness and Piezo1 may affect the morphology of chondrocyte primary cilia by regulating HDAC6 activity.

HDAC6 inhibition regulates substrate stiffness-mediated inflammation signaling in chondrocytes.

Osteoarthritis (OA) is a chronic disease and is difficult to cure. Chondrocytes are highly mechanosensitive. Therefore, mechanical therapies have received attention as a therapeutic direction for OA. The stiffness, as a critical cue of the extracellular matrix (ECM), affects cell growth, development, and death. In this study, we use polydimethylsiloxane (PDMS) to create substrates with varying stiffness for chondrocyte growth, interleukin-1ß (IL-1ß) treatment to mimic the inflammatory environment, and Tubastatin A (Tub A) to inhibit histone deacetylase 6 (HDAC6). Our results show that stiff substrates can be anti-inflammatory and provide a better matrix environment than soft substrates. Inhibition of HDAC6 improves the inflammatory environment caused by IL-1ß and coordinates with inflammation to spread the chondrocyte area and primary cilia elongation. Without IL-1ß and Tub A treatments, the length of the primary cilia rather than frequency is stiffness-dependent, and their length on stiff substrates are greater than that on soft substrates. In conclusion, we demonstrate that stiff substrates, inflammation, and inhibition of HDAC6 enhance the mechanosensitivity of primary cilia and mediate substrate stiffness to suppress inflammation and protect the matrix.

[Matrix stiffening related lncRNA SNHG8 regulates chemosensitivity of ovarian cancer].

Extracellular matrix (ECM) has been implicated in tumor progress and chemosensitivity. Ovarian cancer brings a great threat to the health of women with a significant feature of high mortality and poor prognosis. However, the potential significance of matrix stiffness in the pattern of long non-coding RNAs (lncRNAs) expression and ovarian cancer drug sensitivity is still largely unkown. Here, based on RNA-seq data of ovarian cancer cell cultured on substrates with different stiffness, we found that a great amount of lncRNAs were upregulated in stiff group, whereas SNHG8 was significantly downregulated, which was further verified in ovarian cancer cells cultured on polydimethylsiloxane (PDMS) hydrogel. Knockdown of SNHG8 led to an impaired efficiency of homologous repair, and decreased cellular sensitivity to both etoposide and cisplatin. Meanwhile, the results of the GEPIA analysis indicated that the expression of SNHG8 was significantly decreased in ovarian cancer tissues, which was negatively correlated with the overall survival of patients with ovarian cancer. In conclusion, matrix stiffening related lncRNA SNHG8 is closely related to chemosensitivity and prognosis of ovarian cancer, which might be a novel molecular marker for chemotherapy drug instruction and prognosis prediction.

[Research progress of chondrocyte mechanotransduction mediated by TRPV4 and PIEZOs].

Mechanical signal transduction are crucial for chondrocyte in response to mechanical cues during the growth, development and osteoarthritis (OA) of articular cartilage. Extracellular matrix (ECM) turnover regulates the matrix mechanical microenvironment of chondrocytes. Thus, understanding the mechanotransduction mechanisms during chondrocyte sensing the matrix mechanical microenvironment can develop effective targeted therapy for OA. In recent decades, growing evidences are rapidly advancing our understanding of the mechanical force-dependent cartilage remodeling and injury responses mediated by TRPV4 and PIEZOs. In this review, we highlighted the mechanosensing mechanism mediated by TRPV4 and PIEZOs during chondrocytes sensing mechanical microenvironment of the ECM. Additionally, the latest progress in the regulation of OA by inflammatory signals mediated by TRPV4 and PIEZOs was also introduced. These recent insights provide the potential mechanotheraputic strategies to target these channels and prevent cartilage degeneration associated with OA. This review will shed light on the pathogenesis of articular cartilage, searching clinical targeted therapies, and designing cell-induced biomaterials.

The potential role of mechanosensitive ion channels in substrate stiffness-regulated Ca2+ response in chondrocytes.

PURPOSE: The stiffness of the pericellular matrix (PCM) decreases in the most common degenerative joint disease, osteoarthritis (OA). This study was undertaken to explore the potential functional role of transient receptor potential vanilloid 4 (TRPV4), Piezo1, and Piezo2 in transducing different PCM stiffness in chondrocytes. METHODS AND RESULTS: Polydimethylsiloxane (PDMS) substrates with different stiffness (designated 197 kPa, 78 kPa, 54 kPa, or 2 kPa, respectively) were first prepared to simulate the decrease in stiffness of the PCM that chondrocytes encounter in osteoarthritic cartilage. Next, the TRPV4-, Piezo1-, or Piezo2-knockdown primary chondrocytes (designated TRPV4-KD, Piezo1-KD, or Piezo2-KD cells) were seeded onto these different PDMS substrates. Then, using a Ca2+-imaging system, substrate stiffness-regulated intracellular Ca2+ influx ([Ca2+]i) in chondrocytes was examined to investigate the role of TRPV4, Piezo1, and Piezo2 in Ca2+ signaling in response to different stiffness. Results showed that the characteristics of intracellular [Ca2+]i in chondrocytes regulated by PDMS substrate exhibited stiffness-dependent differences. Additionally, stiffness-evoked [Ca2+]i changes were suppressed in TRPV4-KD, Piezo1-KD, or Piezo2-KD cells compared with control siRNA-treated cells, implying that any channel is fundamental for Ca2+ signaling induced by substrate stiffness. Furthermore, TRPV4-mediated Ca2+ signaling played a central role in the response of chondrocytes to 197 kPa and 78 kPa substrate, while Piezo1/2-mediated Ca2+ signaling played a central role in the response of chondrocytes to 54 kPa and 2 kPa substrate. CONCLUSIONS: Collectively, these findings indicate that chondrocytes might perceive and distinguish the different PCM stiffness by using different mechanosensitive ion channels.

Substrate stiffness-dependent regulatory volume decrease and calcium signaling in chondrocytes.

The pericellular matrix stiffness is strongly associated with its biochemical and structural changes during the aging and osteoarthritis progress of articular cartilage. However, how substrate stiffness modulates the chondrocyte regulatory volume decrease (RVD) and calcium signaling in chondrocytes remains unknown. This study aims to investigate the effects of substrate stiffness on the chondrocyte RVD and calcium signaling by recapitulating the physiologically relevant substrate stiffness. Our results showed that substrate stiffness induces completely different dynamical deformations between the cell swelling and recovering progresses. Chondrocytes swell faster on the soft substrate but recovers slower than the stiff substrate during the RVD response induced by the hypo-osmotic challenge. We found that stiff substrate enhances the cytosolic Ca oscillation of chondrocytes in the iso-osmotic medium. Furthermore, chondrocytes exhibit a distinctive cytosolic Ca oscillation during the RVD response. Soft substrate significantly improves the Ca oscillation in the cell swelling process whereas stiff substrate enhances the cytosolic Ca oscillation in the cell recovering process. Our work also suggests that the TRPV4 channel is involved in the chondrocyte sensing substrate stiffness by mediating Ca signaling in a stiffness-dependent manner. This helps to understand a previously unidentified relationship between substrate stiffness and RVD response under the hypo-osmotic challenge. A better understanding of substrate stiffness regulating chondrocyte volume and calcium signaling will aid the development of novel cell-instructive biomaterial to restore cellular functions.

Mechanical Properties of Chondrocytes Estimated from Different Models of Micropipette Aspiration.

In this study, two viscoelastic creep expressions for the aspirated length of individual solid-like cells undergoing micropipette aspiration (MPA) were derived based on our previous studies wherein the cell size relative to the micropipette and the cell compressibility were taken into account. Next, three mechanical models of MPA, the half-space model (HSM), incompressible sphere model (ICSM), and compressible sphere model (CSM), were employed to fit the MPA data of chondrocytes. The results indicated that the elastic moduli and viscoelastic parameters of chondrocytes for the ICSM and CSM exhibited significantly higher values than those from the HSM (p < 0.001) because of the considerations of the geometric parameter (ξ) and the compressibility of the cell (ν). For the normal chondrocytes, the elastic moduli obtained from the ICSM and CSM (ν = 0.3) were 47.4 and 78.9% higher than those from the HSM. In the viscoelasticity, the parameters k1, k2, and µ for the ICSM were respectively increased by 37.8, 37.9, and 39.0% compared to those from the HSM, whereas for the CSM (ν = 0.3), the above parameters were 135, 314, and 257% higher compared to those from the HSM. And with the increase of ξ and ν, the above mechanical parameters decreased. Furthermore, the thresholds of ξ varying with ν were obtained for the given values of relative errors caused by the HSM in the elastic and viscoelastic parameters. The above findings obviously indicated that the geometric parameter of MPA and the Poisson's ratio of a cell have marked influences on the determination of cellular mechanical parameters by MPA and thus should be considered in the pursuit of more accurate investigations of the mechanical properties of cells.

The effect of matrix stiffness on biomechanical properties of chondrocytes.

The behavior of chondrocytes is regulated by multiple mechanical microenvironmental cues. During development and degenerative disease of articular cartilage, as an external signal, the extracellular matrix stiffness of chondrocytes changes significantly, but whether and how this biophysical cue affects biomechanical properties of chondrocytes remain elusive. In the present study, we designed supporting-biomaterials as  mimics of native pericellular matrix to study the effect of matrix stiffness on chondrocyte morphology and F-actin distribution. Furthermore, the active mechanical behavior of chondrocytes during sensing and responding to different matrix stiffness was quantitatively investigated using atom force microscope technique and theoretical model. Our results indicated that stiffer matrix tends to increase the cell spreading area, the percentage of irregular cell shape distribution and mechanical parameters including elastic modulus (Eelastic), instantaneous modulus (E0), relaxed modulus (ER) and apparent viscosity (µ) of chondrocytes. Knowledge of matrix stiffness-dependent biomechanical behaviors of chondrocytes has important implications for optimizing matrix material and advancing chondrocyte-based applications for functional tissue engineering.

Carbon monoxide augments electrical signaling in cultured neural networks of hippocampal neurons partly through activation of BKCa channels.

Carbon monoxide (CO) is often viewed as a lethal gas in light of its capacity to prevent oxygen uptake in hemoglobin; however, it also functions to regulate a variety of proteins and physiological processes. Here we show that CO is an important chemical cue, to which neurons respond strongly, and this response is then integrated into neural network activity. In cultured mouse hippocampal neurons, CO enhanced synchronized spontaneous cytosolic Ca(2+) oscillations which arose from periodic action potentials through synaptic transmission. We used single-cell patch-clamp recording to investigate the neural network. Our results showed that the frequency of spontaneous and miniature post synaptic current was increased in neurons cultured for 14-18 days after addition of CO, with no change in current amplitude. BK channels have recently been demonstrated to be important in the action of CO. Our results showed that the effect of CO on neural network electrical activity was partly abolished after blocking the BK channels. Altogether, our results suggest that CO can influence neural network electrical activity and that BK channels participate in this regulation process.

Development of alginate-collagen interpenetrating network for osteoarthritic cartilage by in situ softening.

This study presents an alginate-collagen interpenetrating network (IPN) matrix of incorporating collagen fibrils into an alginate hydrogel by physical mixing and controlled gelation. The resulting matrix closely mimics the physiological and pathological stiffness range of the chondrocyte pericellular matrix (PCM). Chondrocytes were cultured within three-dimensional (3D) alginate-collagen IPN matrices with varying stiffness, namely Firm, Medium, and Soft. Alginate lyase was introduced to study the effects of the changes in stiffness of the Firm on chondrocyte response by in situ softening. The developed alginate-collagen IPN matrix displayed good cell-biocompatibility. Compared with stiffer tissue culture plastic (TCP), chondrocytes grown within Firm displayed a stabilized differentiated phenotype characterized by higher expression levels of aggrecan, collagen II, and SOX-9. Moreover, the developed alginate-collagen IPN matrix exhibited a gradually increased percentage of propidium iodide (PI)-positive dead cells with decreasing stiffness. Softer matrices directed cells towards higher proliferation rates and spherical morphologies while stimulating chondrocyte cluster formation. Furthermore, reducing Firm stiffness by in situ softening decreased aggrecan expression, contributing to matrix degradation similar to that seen in osteoarthritis (OA). Hence, the 3D alginate-collagen IPN constructs hold significant potential for in vitro replicating PCM stiffness changes observed in OA cartilage.

Primary cilium-mediated mechanotransduction in cartilage chondrocytes.

Osteoarthritis (OA) is one of the most prevalent joint disorders associated with the degradation of articular cartilage and an abnormal mechanical microenvironment. Mechanical stimuli, including compression, shear stress, stretching strain, osmotic challenge, and the physical properties of the matrix microenvironment, play pivotal roles in the tissue homeostasis of articular cartilage. The primary cilium, as a mechanosensory and chemosensory organelle, is important for detecting and transmitting both mechanical and biochemical signals in chondrocytes within the matrix microenvironment. Growing evidence indicates that primary cilia are critical for chondrocytes signaling transduction and the matrix homeostasis of articular cartilage. Furthermore, the ability of primary cilium to regulate cellular signaling is dynamic and dependent on the cellular matrix microenvironment. In the current review, we aim to elucidate the key mechanisms by which primary cilia mediate chondrocytes sensing and responding to the matrix mechanical microenvironment. This might have potential therapeutic applications in injuries and OA-associated degeneration of articular cartilage.

TRPV4 and PIEZO Channels Mediate the Mechanosensing of Chondrocytes to the Biomechanical Microenvironment.

Articular cartilage and their chondrocytes are physiologically submitted to diverse types of mechanical cues. Chondrocytes produce and maintain the cartilage by sensing and responding to changing mechanical loads. TRPV4 and PIEZOs, activated by mechanical cues, are important mechanosensing molecules of chondrocytes and have pivotal roles in articular cartilage during health and disease. The objective of this review is to introduce the recent progress indicating that the mechanosensitive ion channels, TRPV4 and PIEZOs, are involved in the chondrocyte sensing of mechanical and inflammatory cues. We present a focus on the important role of TRPV4 and PIEZOs in the mechanotransduction regulating diverse chondrocyte functions in the biomechanical microenvironment. The review synthesizes the most recent advances in our understanding of how mechanical stimuli affect various cellular behaviors and functions through differentially activating TRPV4 and PIEZO ion channels in chondrocyte. Advances in understanding the complex roles of TRPV4/PIEZO-mediated mechanosignaling mechanisms have the potential to recapitulate physiological biomechanical microenvironments and design cell-instructive biomaterials for cartilage tissue engineering.

The study of mechanical and drug release properties of the mineralized collagen/polylactic acid scaffold by tuning the crystalline structure of polylactic acid.

Open bone fractures in clinical are not only difficult to heal but also at a high risk of infections. Annual cases of fractures which result from osteoporosis amount to approximately 9 million. The objective of this study is to load the antibiotic drug of vancomycin and tune its controlled delivery on a bone repair scaffold material of Mineralized Collagen/poly(lactic acid) (MCP) via changing the crystallinity of poly(lactic acid) to achieve inhibiting infection while repairing defects. We explored the crystallization process of the material during molding and prepared non-crystalline MCP1, MCP2, MCP3 and MCP4 by rapid freeze forming and crystalline MCP5 by tuning temperature decreasing rate. This method can control the micropore structure of the material; and the material changes from brittleness to toughness, which greatly enhances the control of mechanical properties. The drug release behavior of the material was studied for 28 days. Furthermore, the antibacterial property of the material was tested by the zone of inhibition, which shows the material good bacteriostasis. The controllable MCPs are expected to be substitutes for the treatment of infectious bone defects applying to clinical practical treatment.

The effect of calcium phosphate and silk fibroin nanofiber tuning on properties of calcium sulfate bone cements.

Calcium sulfate (CS) bone cements have been used as bone substitutes for a long time, but their clinical use is currently limited due to their rapid degradation rate and brittleness. This work aimed to study the effect of α-tricalcium phosphate (α-TCP) and silk fibroin nanofibers (SFF) on CS bone cements. The bone cements were prepared from α-CS hemihydrate (α-CSH), calcium sulfate dihydrate (CSD; as a setting accelerator) and varying α-TCP contents (0%, 5%, 10%, 15%, 20% and 25%), with SFF solution or deionized water as the solidification solution at the same liquid/solid ratio. Scanning electron microscopy, particle size distribution, x-ray diffraction and Fourier transform infrared spectroscopy were used to measure the composition and characterize the properties of the materials. The compressive strength, setting time and weight loss rate of samples were also tested. Cytotoxicity was evaluated by a Cell Counting Kit-8 assay. The results suggest that the tuning of α-TCP and SFF has an important role in determining the compressive strength and degradation rate of CS bone cements, and the properties could be changed by varying the content of α-TCP. Moreover, cell experiments showed no toxicity of the samples towards MC3T3 cells. Thus, the materials prepared from α-CSH, CSD, α-TCP and SFF in this work could provide the basis for research into CS-based bone repair materials.

Microniche geometry modulates the mechanical properties and calcium signaling of chondrocytes.

In articular cartilage, the function of chondrocytes is strongly related to their zone-specific microniche geometry defined by pericellular matrix. Microniche geometry is critical for regulating the phenotype and function of the chondrocyte in native cartilage and tissue engineering constructs. However the role of microniche geometry in the mechanical properties and calcium signaling of chondrocytes remains unknown. To recapitulate microniche geometry at single-cell level, we engineered three basic physiological-related polydimethylsiloxane (PDMS) microniches geometries fabricated using soft lithography. We cultured chondrocytes in these microniche geometries and quantified cell mechanical properties using atomic force microscopy (AFM). Fluorescent calcium indicator was used to record and quantify cytosolic Ca2+ oscillation of chondrocytes in different geometries. Our work showed that microniche geometry modulated the mechanical behavior and calcium signaling of chondrocytes. The ellipsoidal microniches significantly enhanced the mechanical properties of chondrocytes compared to spheroidal microniche. Additionally, ellipsoidal microniches can markedly improved the amplitude but weakened the frequency of cytosolic Ca2+ oscillation in chondrocytes than spheroidal microniche. Our work might reveal a novel understanding of chondrocyte mechanotransduction and therefore be useful for designing cell-instructive scaffolds for functional cartilage tissue engineering.

[Age-related biomechanical properties of chondrocytes in rabbit knee articular cartilage].

OBJECTIVE: To fully demonstrate the alterations about the viscoelastic properties of chondrocytes in rabbit knee articular cartilage. METHODS: Fifteen New Zealand white rabbits were divided into 3 age groups: young group (1 month), adult group (8 months) and old group (31 months). All rabbits were sacrificed and isolated from knee joint and digested into chondrocytes. The micropipette aspiration combined with Half-space model was used to quantify changes in viscoelastic properties of chondrocytes. RESULTS: Experimental studies have shown that the changes of aspiration length with time in old group were obviously different from young group and adult group. But similar variations were found between the latter two groups. The viscoelastic properties of chondrocytes in old group exhibited a significantly lower instantaneous modulus E0 (0.55 +/- 0.05 kPa), equilibrium modulus E infinity (0.28 +/- 0.04 kPa) and apparent viscosity micro (4.10 +/- 0.61 kPa x s) as compared with young group (0.67 +/- 0.10), (0.37 +/- 0.09), (6.29 +/- 0.92) kPa x s (P < 0.001) versus adult group: (0.65 +/- 0.07), (0.35 +/- 0.05), (6.01 +/- 0.89) kPa x s (P < 0.001). But no difference were found between the latter two groups (P > 0.05). CONCLUSION: In response to a step pressure, chondrocytes in each group exhibited the viscoelastic solid creep behavior. The viscoelastic properties of chondrocytes markedly decreased in old group.

Calcium sulfate bone cements with nanoscaled silk fibroin as inducer.

Both nanostructures and conformations of different protein/polysaccharide additives have critical influence on the performance of calcium sulfate (CS) bone cements. Silk fibroin (SF) as matrix and additives has been introduced to develop bone scaffolds and cements. Here, ß-sheet-rich SF nanofibers (SFF) was used to tune the solidification of CS, achieving better mechanical and biological properties. The ratio of SFF was adjusted to further optimize CS functions. Compared to that regulated with natural silk fibers (NSF) and SF solutions (SFS), the SFF-induced CS showed smaller size and more filament structures. Better mechanical properties were achieved, suggesting the superiority of the SFF as the solidifying solution to combine with α-calcium sulfate hemihydrate (α-CSH) at the same liquid/solid (L/S) ratio. Scanning electron microscope, X-ray diffraction, Fourier transform infrared spectroscopy, setting time, porosity, mechanical performance test, degradation performance test, and water resistance test were used to demonstrate the properties of this bone repair cement. Cell culture experiments in vitro was used to evaluate the biocompatibility of this composited material. In conclusion, the results demonstrated that nanofibers was a better form of SF in the modification of CSH cement. And the research conducted in this article on improving the mechanical and biological properties of CSH would supported the reference for later clinical experiments. © 2019 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater 107B:2611-2619, 2019.

An experimental study on collagen content and biomechanical properties of sclera after posterior sclera reinforcement.

BACKGROUND: The development of pathological myopia is associated with reduced scleral collagen accumulation, scleral thinning, and loss of scleral tissue, in both humans and animal models. Posterior scleral reinforcement (PSR) was considered as an effective way for treating pathological myopia. Yet it is not well understood the possible role of collagen on the sclera reinforcement mechanisms in the PSR surgery. METHODS: PSR surgery was performed on the normal adult New Zealand white rabbits eyes. Human sclera was used as reinforcement materials. At 1, 2, 3, 6, 9 months after the PSR surgery, scleral hydroxyproline (Hyp) synthesis and collagen fibers arrangement were determined by enzymolysic hydrolysis assay and histological morphology technique. An Instron test machine was used to investigate the elastic modulus of sclera. FINDINGS: It was found that the elastic modulus and Hyp content of reinforced sclera were lower at first month after surgery, and then gradually up to physiological level in the following months. Those two indexes were close to that of the normal control groups at 9 months. INTERPRETATION: These findings indicate that sclera elastic modulus was associated with both change of Hyp content and collagen fibers arrangement after PSR. The therapeutic effect of PSR surgery was confirmed not only from biological but also biomechanical aspects.

A study of the initial adhesive force of cells on silk fibroin-based materials using micropipette aspiration.

With the development of biomaterials, more attention is paid to the adhesion characteristics between cells and materials. It is necessary to study the adhesive force with a suitable method. Silk fibroin (SF) is widely investigated in biomedical application due to its novel biocompatibility and mechanical properties. In this article, the micropipette aspiration method and measurement pattern of uniform cells in round shape (UCR) was used to study the initial adhesive force of three types of cells on pure silk fibroin films (SFFs). We also compared the adhesive forces of modified SFFs with that of pure SFFs. The results of adhesive force in the initial adhesive stage were in concordance with the results of MTT assay and microscope observation, which were confirmed by the above three cell lines and four kinds of SFFs. The results indicated UCR was an efficient and quantitative measurement pattern in initial adhesion stage. This article also provides a useful method in identifying initial cell-materials interactions.

[Mechanical properties of chondrocytes isolated from normal articular cartilage: experiment with rabbit knees].

OBJECTIVE: To measure the mechanical properties of chondrocytes of articular cartilage of knee in order to provide relevant technical parameters into the development of cartilage tissue engineering. METHODS: Eight NZW rabbits were killed, and their bilateral knee joints were taken out. The articular cartilage of the left knees were used for histological study, and articular cartilage of the right knees was digested with 0.4% pronase and 0.025% collagenase type II so as to make isolation of chondrocytes. The viability rate of the isolated chondrocytes was detected by trypan blue staining. The diameters of the chondrocytes in the histological sections and single cell suspension were measured respectively. The mechanical properties of the chondrocytes were determined using micropipette aspiration technique coupled with half-space model. RESULTS: In the normal articular cartilage 4 structural zones were differentiated: tangential, transitional, radial, and calcified zones. The viability rate of chondrocytes was 98.2% on average after isolation. The mean diameter of the chondrocytes in the histological sections was (14.2+/-2.9) microm, not significantly different from that in the single cell suspension [(14.9+/-2.2) microm, t=1.31, P=0.19]. In response to a prescribed pressure, the chondrocytes exhibited viscoelastic solid creep behavior characterized initially by a jump in displacement followed by a monotonically decreasing rate of deformation that generally reached an equilibrium displacement. The cells were observed to deform to a length of as much as three times the radius of the micropipette without completely entering the micropipette. The cells were then extruded from the micropipette and completely recovered. The Young's modulus of the normal chondrocytes was (0.57+/-0.43) kPa, and the k1, k2, and micro of the viscoelastic parameters were (0.37+/-0.07) kPa, (0.29+/-0.04) kPa, and (6.36+/-1.12) kPa-s respectively. The k1 is positively correlated to the cell diameter (r=0.4, P=0.031). CONCLUSION: Chondrocytes from normal articular cartilage behave as a viscoelastic solid. Micropipette aspiration technique is an efficient method for the study of chondrocyte biomechanics.