Anti-adhesive effect of chondrocyte-derived extracellular matrix surface-modified with poly-L-lysine.

After an injury, soft tissue structures in the body undergo a natural healing process through specific phases of healing. Adhesions occur as abnormal attachments between tissues and organs through the formation of blood vessels and/or fibrinous adhesions during the regenerative repair process. In this study, we developed an adhesion-preventing membrane with an improved physical protection function by modifying the surface of chondrocyte-derived extracellular matrices (CECM) with anti-adhesion function. We attempted to change the negative charge of the CECM surface to neutral using poly-L-lysine (PLL) and investigated whether it blocked fibroblast adhesion to it and showed an improved anti-adhesion effect in animal models of tissue adhesion. The surface of the membrane was modified with PLL coating (PLL 10), which neutralized the surface charge. We confirmed that the surface characteristics except for the potential difference were maintained after the modification and tested cell attachment in vitro. Adhesion inhibition was identified in a peritoneal adhesion animal model at 1 week and in a subcutaneous adhesion model for 4 weeks. Neutralized CECM (N-CECM) suppressed fibroblast and endothelial cell adhesion in vitro and inhibited abdominal adhesions in vivo. The CECM appeared to actively inhibit the infiltration of endothelial cells into the injured site, thereby suppressing adhesion formation, which differed from conventional adhesion barriers in the mode of action. Furthermore, the N-CECM remained intact without degradation for more than 4 weeks in vivo and exerted anti-adhesion effects for a long time. This study demonstrated that PLL10 surface modification rendered a neutral charge to the polymer on the extracellular matrix surface, thereby inhibiting cell and tissue adhesion. Furthermore, this study suggests a means to modify extracellular matrix surfaces to meet the specific requirements of the target tissue in preventing post-surgical adhesions.

The effect of distance between holes on the structural stability of subchondral bone in microfracture surgery: a finite element model study.

BACKGROUND: Microfracture is a surgical technique that involves creating multiple holes of 3-4 mm depth in the subchondral bone to recruit stem cells in the bone marrow to the lesion, inducing fibrocartilage repair and knee cartilage regeneration. Recently, it has been reported that increasing the exposed area of the lower cartilaginous bone (drilling a lot of holes) increases the outflow of stem cells, which is expected to affect the physical properties of the subchondral bone when the exposed area is large. The purpose of this study was to analyse the effect of the distance between the holes in the microfracture procedure on the structural stability of the osteochondral bone using a finite element method. METHODS: In this study, lateral aspects of the femoral knee, which were removed during total knee arthroplasty were photographed using microtomography. The model was implemented using a solitary walks program, which is a three-dimensional simplified geometric representation based on the basic microtomography data. A microfracture model was created by drilling 4 mm-deep holes at 1, 1.5, 2, 2.5, 3, 4, and 5 mm intervals in a simplified three-dimensional (3D) geometric femoral model. The structural stability of these models was analysed with the ABAQUS program. We compared the finite element model (FEM) based on the microtomography image and the simplified geometric finite element model. RESULTS: Von Mises stress of the subchondral bone plate barely increased, even when the distance between holes was set to 1 mm. Altering the distance between the holes had little impact on the structural stability of the subchondral bone plate. Safety factors were all below 1. CONCLUSIONS: Although we did not confirm an optimal distance between holes, this study does provide reference data and an epidemiological basis for determining the optimal distance between the holes used in the microfracture procedure.

Phlorofucofuroeckol A from Ecklonia cava ameliorates TGF-ß1-induced fibrotic response of human tracheal fibroblasts via the downregulation of MAPKs and SMAD 2/3 pathways inactivated TGF-ß receptor.

The objective of this study was to investigate inhibitory effects of a bioactive compound isolated from Ecklonia cava on fibrotic responses to transforming growth factor-ß1 (TGF-ß1)-stimulated Hs680. Tr human tracheal fibroblasts and the associated mechanisms of action. Post consecutive purification, a potent bioactive compound was identified phlorofucofuroeckol A. Phlorofucofuroeckol A significantly suppressed protein expression levels of collagen type I and α-smooth muscle actin (α-SMA) on TGF-ß1-stimulated Hs680. Tr human tracheal fibroblasts. Further mechanistic studies determined that phlorofucofuroeckol A suppressed the phosphorylation of p38, extracellular regulated kinase (ERK), and c-Jun N-terminal kinase (JNK) and SMAD 2/3 in TGF-ß1-stimulated Hs680. Tr human tracheal fibroblasts. Moreover, we could show that phlorofucofuroeckol A inhibits binding of TGF-ß1 to its TGF-ß receptor by molecular docking. Based on the results, we propose that phlorofucofuroeckol A suppresses the MAPKs and SMAD 2/3 pathways and relieves cellular fibrotic activities, thus preventing tracheal fibrosis.

Cross-linked electrospun cartilage acellular matrix/poly(caprolactone-co-lactide-co-glycolide) nanofiber as an antiadhesive barrier.

In this work, we chose cartilage acellular matrix (CAM) as a promising antiadhesive material because CAM effectively inhibits the formation of blood vessels, and we used electrospinning to prepare antiadhesive barriers. Additionally, we synthesized N-hydroxysuccinimide (NHS)-poly(caprolactone-co-lactide-co-glycolide)-NHS (MP) copolymers (to tune degradation) as a cross-linking agent for CAM. This is the first report on the development of electrospun cross-linked (Cx) CAM/MP (CA/P) nanofiber (NF) (Cx-CA/P-NF) with a tunable degradation period as an antiadhesive barrier. Compared with the CA/P-NF before cross-linking, the electrospun Cx-CA/P-NF after cross-linking showed different biodegradation. Cx-CA/P-NF significantly inhibited the in vitro attachment and proliferation of human umbilical vein endothelial cells (HUVECs), as confirmed by an MTT assay and scanning electron microscopy images. Cx-CA/P-NFs implanted between a surgically damaged peritoneal wall and cecum gradually degraded in 7 days; this process was monitored by NIR imaging. The in vivo evaluation of the anti-tissue adhesive effect of Cx-CA/P-NFs revealed little adhesion, few blood vessels, and negligible inflammation at 7 days determined by hematoxylin and eosin staining. ED1 staining of Cx-CA/P-NFs showed infiltration of few macrophages because of the inflammatory response to the Cx-CA/P-NF as compared with an untreated injury model. Additionally, Cx-CA/P-NFs significantly suppressed the formation of blood vessels between the peritoneal wall and cecum, according to CD31 staining. Overall, Cx-CA/P-NFs yielded little adhesion, infiltration by macrophages, or formation of blood vessels in a postoperative antiadhesion assay. Thus, it is reasonable to conclude that the Cx-CA/P-NF designed herein successfully works as an antiadhesive barrier with a tunable degradation period. STATEMENT OF SIGNIFICANCE: The cartilage acellular matrix (CAM) can inhibit the formation of fibrous tissue bridges and blood vessels between the tissue at an injured site and the surrounding healthy tissues. However, CAM has not been rigorously investigated as an antiadhesive barrier. In this manuscript, the cross-linked CAM nanofiber (Cx-CA/P-NF) designed herein successfully works as an antiadhesive barrier. Cx-CA/P-NFs yielded little adhesion, infiltration by macrophages, or formation of blood vessels in a postoperative antiadhesion assay. Moreover, we demonstrated the suitable properties of Cx-CA/P-NF such as easy cross-linking by maintaining the antiadhesive properties, controllable biodegradation, and in vivo antiadhesive effect of Cx-CA/P-NF.

Nondestructive Assessment of Glycosaminoglycans in Engineered Cartilages Using Hexabrix-Enhanced Micro-Computed Tomography.

It is very useful to evaluate the content and 3D distribution of extracellular matrix non-destructively in tissue engineering. This study evaluated the feasibility of using micro-computed tomography (µCT) with Hexabrix to measure quantitatively sulfated glycosaminoglycans (GAGs) of engineered cartilage. Rabbit chondrocytes at passage 2 were used to produce artificial cartilages in polyglycolic acid scaffolds in vitro. Engineered cartilages were incubated with Hexabrix 320 for 20 min and analyzed via µCT scanning. The number of voxels in the 2D and 3D scanning images were counted to estimate the amount of sulfated GAGs. The optimal threshold value for quantification was determined by regression analysis. The 2D µCT images of an engineered cartilage showed positive correlation with the histological image of Safranin-O staining. Quantitative data obtained with the 3D µCT images of 14 engineered cartilages showed strong correlation with sulfated GAGs contents obtained by biochemical analysis (R2 = 0.883, p < 0.001). Repeated exposure of engineered cartilages to Hexabrix 320 and µCT scanning did not significantly affect cell viability, total DNA content, or the total content of sulfated GAGs. We conclude that µCT imaging using Hexabrix 320 provides high spatial resolution and sensitivity to assess the content and 3D distribution of sulfated GAGs in engineered cartilages. It is expected to be a valuable tool to evaluate the quality of engineered cartilage for commercial development in the future.

Subchondral bone scan uptake correlates with articular cartilage degeneration in osteoarthritic knees.

AIM: The aim of this study was to analyze subchondral bone scan uptake in osteoarthritic knees with reference to subchondral bone microstructure and articular cartilage histology. METHODS: This cross-sectional, laboratory study evaluated 123 human distal femoral condyle specimens of 67 patients after joint replacement surgery. All patients were preoperatively examined with bone scan of the knee joint. Specimens were evaluated for cartilage histology and micro-computed tomography analysis of subchondral bone. Data between bone scan, histology and micro-computed tomography were statistically analyzed using either coefficient of correlation, Student's t-test or one-way analysis of variance with Tukey post hoc test. RESULTS: Bone scan grading and histological articular cartilage degeneration scores showed significant correlation (r = 0.812, P < 0.001). Both bone scan positive and histologically confirmed osteoarthritis samples showed increase in subchondral trabecular bone volume and thickness, reflected in micro-computed tomography. Overall, positive predictive value (%) of bone scan for osteoarthritic cartilage lesions was 91.9%, and the sensitivity and specificity were 88.3% and 60%, respectively. Histology showed that bone scan has both a high positive predictive and a low negative predictive value for detection of osteoarthritic cartilage lesions. CONCLUSION: Bone scan uptake correlated with articular cartilage degeneration in osteoarthritic knees. Bone scan may be a useful diagnostic tool that reflects pathologic changes of cartilage in osteoarthritis.

Extracellular Matrix (ECM) Multilayer Membrane as a Sustained Releasing Growth Factor Delivery System for rhTGF-ß3 in Articular Cartilage Repair.

Recombinant human transforming growth factor beta-3 (rhTGF-ß3) is a key regulator of chondrogenesis in stem cells and cartilage formation. We have developed a novel drug delivery system that continuously releases rhTGF-ß3 using a multilayered extracellular matrix (ECM) membrane. We hypothesize that the sustained release of rhTGF-ß3 could activate stem cells and result in enhanced repair of cartilage defects. The properties and efficacy of the ECM multilayer-based delivery system (EMLDS) are investigated using rhTGF-ß3 as a candidate drug. The bioactivity of the released rhTGF-ß3 was evaluated through chondrogenic differentiation of mesenchymal stem cells (MSCs) using western blot and circular dichroism (CD) analyses in vitro. The cartilage reparability was evaluated through implanting EMLDS with endogenous and exogenous MSC in both in vivo and ex vivo models, respectively. In the results, the sustained release of rhTGF-ß3 was clearly observed over a prolonged period of time in vitro and the released rhTGF-ß3 maintained its structural stability and biological activity. Successful cartilage repair was also demonstrated when rabbit MSCs were treated with rhTGF-ß3-loaded EMLDS ((+) rhTGF-ß3 EMLDS) in an in vivo model and when rabbit chondrocytes and MSCs were treated in ex vivo models. Therefore, the multilayer ECM membrane could be a useful drug delivery system for cartilage repair.

Comparison of fetal cartilage-derived progenitor cells isolated at different developmental stages in a rat model.

Fetal cartilage-derived progenitor cells (FCPCs) could be a useful cell source in cell-based therapies for cartilage disorders. However, their characteristics can vary depending on the developmental stages. The aim of this study was to compare the characteristics of rat FCPCs from the hind limb on embryonic day 14 (E14), E16 and E20 regarding proliferation, pluripotency, and differentiation. Morphologically, rat fetal cartilage tissue showed an increase in cartilaginous differentiation features (Safranin-O, type II collagen) and decrease in pluripotency marker (Sox2) in the order of E14, E16 and E20. E14 FCPCs showed significantly higher doubling time compared to E16 and E20 FCPCs. While the E14 FCPCs expressed pluripotent genes (Sox2, Oct4, Nanog), the E16 and E20 FCPCs expressed chondrogenic markers (Sox9, Col2a1, Acan). E20 FCPCs showed the highest ability to both chondrogenic and adipogenic differentiation and E14 FCPCs showed relatively better activity in osteogenic differentiation. Further analysis showed that E20 FCPCs expressed both adipogenic (C/ebpß) and osteogenic (Runx2, Sp7, Taz) transcription factors as well as chondrogenic transcription factors. Our results show an inverse relationship overall between the expression of pluripotency genes and that of chondrogenic and lineage-specific genes in FCPCs under development. Due to its exceptional proliferation and chondrogenic differentiation ability, fetal cells from epiphyseal cartilage (E20 in rats) may be a suitable cell source for cartilage regeneration.

In vivo degradation profile of porcine cartilage-derived extracellular matrix powder scaffolds using a non-invasive fluorescence imaging method.

We present a non-invasive fluorescence method for imaging of scaffold degradation in vivo by quantifying the degradation of porcine cartilage-derived extracellular matrix powder (PCP).Three-dimensional porous scaffolds should be biocompatible and bioresorbable, with a controllable degradation and resorption rate to match tissue growth. However, in vivo scaffold degradation and tissue ingrowth processes are not yet fully understood. Unfortunately, current analysis methods require animal sacrifice and scaffold destruction for the quantification of scaffold degradation and cannot monitor the situation in real time. In this study, Cy3, a fluorescent dye, was used for visualizing PCP and a real-time degradation profile was obtained quantitatively by a non-invasive method using an imaging system in which the reduction in fluorescence intensity depended on PCP scaffold degradation. Real-time PCP scaffold degradation was confirmed through changes in the volume and morphology of the scaffold using micro-computed tomography and microscopy. Our results suggest that extracellular matrix degradation was induced by collagen degradation because of the binding between Cy3 and collagen. This non-invasive real-time monitoring system for scaffold degradation will increase our understanding of in vivo matrix and/or scaffold degradation.

Repair of partial thickness cartilage defects using cartilage extracellular matrix membrane-based chondrocyte delivery system in human Ex Vivo model.

Treatment options for partial thickness cartilage defects are limited. The purpose of this study was to evaluate the efficacy of the chondrocyte-seeded cartilage extracellular matrix membrane in repairing partial thickness cartilage defects. First, the potential of the membrane as an effective cell carrier was investigated. Secondly, we have applied the chondrocyte-seeded membrane in an ex vivo, partial thickness defect model to analyze its repair potential. After culture of chondrocytes on the membrane in vitro, cell viability assay, cell seeding yield calculation and cell transfer assay were done. Cell carrying ability of the membrane was also tested by seeding different densities of cells. Partial defects were created on human cartilage tissue explants. Cell-seeded membranes were applied using a modified autologous chondrocyte implantation technique on the defects and implanted subcutaneously in nude mice for 2 and 4 weeks. In vitro data showed cell viability and seeding yield comparable to standard culture dishes. Time dependent cell transfer from the membrane was observed. Membranes supported various densities of cells. Ex vivo data showed hyaline-like cartilage tissue repair, integrated on the defect by 4 weeks. Overall, chondrocyte-seeded cartilage extracellular membranes may be an effective and feasible treatment strategy for the repair of partial thickness cartilage defects.

Preparation of Extracellular Matrix Developed Using Porcine Articular Cartilage and In Vitro Feasibility Study of Porcine Articular Cartilage as an Anti-Adhesive Film.

In this study, we examined whether porcine articular cartilage (PAC) is a suitable and effective anti-adhesive material. PAC, which contained no non-collagenous tissue components, was collected by mechanical manipulation and decellularization of porcine knee cartilage. The PAC film for use as an anti-adhesive barrier was easily shaped into various sizes using homemade silicone molds. The PAC film was cross-linked to study the usefulness of the anti-adhesive barrier shape. The cross-linked PAC (Cx-PAC) film showed more stable physical properties over extended periods compared to uncross-linked PAC (UnCx-PAC) film. To control the mechanical properties, Cx-PAC film was thermally treated at 45 °C or 65 °C followed by incubation at room temperature. The Cx-PAC films exhibited varying enthalpies, ultimate tensile strength values, and contact angles before and after thermal treatment and after incubation at room temperature. Next, to examine the anti-adhesive properties, human umbilical vein endothelial cells (HUVECs) were cultured on Cx-PAC and thermal-treated Cx-PAC films. Scanning electron microscopy, fluorescence, and MTT assays showed that HUVECs were well adhered to the surface of the plate and proliferated, indicating no inhibition of the attachment and proliferation of HUVECs. In contrast, Cx-PAC and thermal-treated Cx-PAC exhibited little and/or no cell attachment and proliferation because of the inhibition effect on HUVECs. In conclusion, we successfully developed a Cx-PAC film with controllable mechanical properties that can be used as an anti-adhesive barrier.

The Degeneration of Meniscus Roots Is Accompanied by Fibrocartilage Formation, Which May Precede Meniscus Root Tears in Osteoarthritic Knees.

BACKGROUND: Fibrocartilage metaplasia in tendons and ligaments is an adaptation to compression as well as a pathological feature during degeneration. Medial meniscus posterior roots are unique ligaments that resist multidirectional forces, including compression. PURPOSE: To characterize the degeneration of medial meniscus posterior root tears in osteoarthritic knees, with an emphasis on fibrocartilage and calcification. STUDY DESIGN: Cross-sectional study; Level of evidence, 3. METHODS: Samples of medial meniscus posterior roots were harvested from cadaveric specimens and patients during knee replacement surgery and grouped as follows: normal reference, no tear, partial tear, and complete tear. Degeneration was analyzed with histology, immunohistochemistry, and real-time polymerase chain reaction. Uniaxial tensile tests were performed on specimens with and without fibrocartilage. Quantifiable data were statistically analyzed by the Kruskal-Wallis test with the Dunn comparison test. RESULTS: Thirty, 28, and 42 samples harvested from 99 patients were allocated into the no tear, partial tear, and complete tear groups, respectively. Mean modified Bonar tendinopathy scores for each group were 3.97, 9.31, and 14.15, respectively, showing a higher degree of degeneration associated with the extent of the tear (P < .05 for all groups). The characterization of root matrices revealed an increase in fibrocartilage according to the extent of the tear. Tear margins revealed fibrocartilage in 59.3% of partial tear samples and 76.2% of complete tear samples, with a distinctive cleavage-like shape. Root tears with a similar shape were induced within fibrocartilaginous areas during uniaxial tensile testing. Even in the no tear group, 56.7% of samples showed fibrocartilage in the anterior margin of the root, adjacent to the meniscus. An increased stained area of calcification and expression of the ectonucleotide pyrophosphatase/phosphodiesterase 1 gene were observed in the complete tear group compared with the no tear group (P < .0001 and P = .24, respectively). CONCLUSION: Fibrocartilage and calcification increased in medial meniscus posterior roots, associated with the degree of the tear. Both findings, which impair the ligament's resistance to tension, may play a pivotal role during the pathogenesis of degenerative meniscus root tears in osteoarthritic knees. Fibrocartilage and calcification may be useful as diagnostic markers as well as markers of degeneration, which may aid in determining the treatment modality in meniscus root tears. The presence of fibrocartilage in intact roots may suggest an impending tear in osteoarthritic knees.

Histomorphochemical comparison of microfracture as a first-line and a salvage procedure: is microfracture still a viable option for knee cartilage repair in a salvage situation?

Microfracture is considered as the first-line procedure for knee cartilage repair, but the results of microfracture seem less predictable and rather controversial in a salvage situation. Thus, the purpose of the study was to histomorphochemically compare microfracture as a salvage procedure with microfracture as a first-line procedure in a rabbit model. We hypothesized that microfracture in a salvage situation would result in histomorphochemically inferior cartilage repair compared to microfracture as a first-line procedure, and the inferiority would be attributed to less migration of reparable marrow cells to the defect due to destruction of microarchitecture of the subchondral bone. Thirty-six New Zealand white rabbits were divided into three groups: (i) untreated full-thickness chondral defect, (ii) single microfracture treatment (first microfracture group), and (iii) repeated microfracture in 8 weeks after the first procedure (second microfracture group). In each group, rabbits were sacrificed at the end of 8 weeks, and osteochondral specimens at the repair sites were obtained for histomorphochemical analysis. Results showed that microfracture as a salvage procedure resulted in overall inferior cartilage repair histomorphochemically compared with microfracture as a first-line procedure, which correlated with deteriorative changes in the quality of underlying subchondral bone rather than intrinsic incapability to recruit the reparative cells in the defect area. In conclusion, although a comparable number of reparable cells and a mechanically weakened subchondral bone are anticipated, more study is necessary to clearly determine when a microfracture should be performed in a situation.

Injectable extracellular matrix hydrogel developed using porcine articular cartilage.

This work was first development of a delivery system capable of maintaining a sustained release of protein drugs at specific sites by using potentially biocompatible porcine articular cartilage. The prepared porcine articular cartilage powder (PCP) was easily soluble in phosphate-buffered saline. The PCP suspension easily entrapped bovine serum albumin-fluorescein isothiocyanate (BSA-FITC) in pharmaceutical formulations at room temperature. The aggregation of PCP and BSA-FITC was confirmed by dynamic light scattering. When the BSA-FITC-loaded PCP suspension was subcutaneously injected into rats, it gelled and formed an interconnecting three-dimensional PCP structure that allowed BSA to penetrate through it. The amount of BSA-FITC released from the PCP hydrogel was determined in rat plasma and monitored by real-time in vivo molecular imaging. The data indicated sustained release of BSA-FITC for 20 days in vivo. In addition, the PCP hydrogel induced a slight inflammatory response. In conclusion, we showed that the PCP hydrogel could serve as a minimally invasive therapeutics depot.

Effect of different bone marrow stimulation techniques (BSTs) on MSCs mobilization.

The therapeutic effect of bone marrow stimulation techniques (BSTs) is mainly attributed to the role of mesenchymal stem cells (MSCs) from the bone marrow. However, no studies have directly shown the amount of MSCs drained by BSTs. This study hypothesized that differences in the opening of subchondral bone affect the number of MSCs drained from the bone marrow. We purposed that as the exposed area and hole size of BSTs vary, the number of MSCs drained out was measured. Three groups of different BSTs were designed that have variations in the sizes of total exposed area and individual holes. Three different BSTs using a curette, 1.5- and 0.8-mm awls were carried out on the full-thickness femoral cartilage defect of young rabbits. After BST, the number of MSCs in the blood clot was measured by CFU-Fs assay. As the size of the total exposed area increased, so did the number of MSCs obtained. The number of MSCs drained from bone marrow may vary depending on different BSTs and this could affect therapeutic efficacy of cartilage defect. As current microfracture (MF) method cannot drain the most MSCs clinically, more improved surgery technique is needed.

Manufacturing of multi-layered nanofibrous structures composed of polyurethane and poly(ethylene oxide) as potential blood vessel scaffolds.

One of the current limitations in using electrospun nanofibrous materials for tissue engineering is that cells have difficulty penetrating into the materials. For this, multi-layered electrospun structures composed of polyurethane (PU) and poly(ethylene oxide) (PEO) were fabricated and tested in vitro. A 20% (w/v) PU solution was electrospun for 30 min, while a 20% (w/v) PEO solution was electrospun for 5, 15 or 30 min, alternatively. Then, the PEO was extracted by immersing the structure in distilled water to make multi-layered structure. The characteristics of fabricated structures were examined by SEM, FT-IR spectroscopy, mechanical tests and cell penetration test. The bioactivities of smooth muscle cells (SMCs) on these scaffolds were assessed by quantifying DNA, collagen and glycosaminoglycan (GAG) levels. Although hybrid PEO-extracted scaffolds had a little of residual PEO, they were more penetrable than PU alone scaffolds. Also, they showed higher bioactivity than PU-alone scaffolds. The results of this study provided potential of this structure in the application not only to the development of artificial blood vessels but also to other types for tissue engineering.

Effects of intermittent hydrostatic pressure on cell adhesive forces and other related parameters under various resting periods.

The purpose of this study is to investigate the effects of intermittent hydrostatic pressure with various resting periods on the cell adhesive forces and other parameters related to spreading in early stage of cell adhesion. For this, bovine pulmonary arterial endothelium (CPAE, cell line), porcine articular chondrocytes, and human endothelial cells (HECs) were used. The cells were divided into six different experimental groups. Control group was cultured without stimulation, while the constant pressure was applied to group 1 for 2 h. Groups 2-5 were intermittently pressurized for 2 min at a time over a 2-h period with 5, 10, 15, and 20-min resting periods, respectively. Each group was then split into two subgroups, depending whether it experienced extra 60 min stabilization period after stimulation. The average adhesive force and the number and area of focal contacts were significantly higher in the group 4 subgroup, which received an extra 60 min of culture than in the other groups. Similarly, other parameters in this subgroup were significantly different from those in the other groups. The focal contact area and adhesive force were closely related (r = 0.990). We concluded that the mechanical stimuli affect cell adhesion and that the length of the resting period influences the adhesive forces generated at the early stages of adhesion.

Effects of mechanical stimuli and microfiber-based substrate on neurite outgrowth and guidance.

We introduced mechanical stimuli and micropatterned substrate with microfibers to investigate their effects on neurite outgrowth along with nerve growth factor in vitro. Two types of surface morphology were used: a surface that was coated by laminin alone and a surface where in microfibers was added on the laminin surface. PC-12 cells were seeded on both surface types and cultured for 2 d. The magnitudes of shear stresses ranged from 0.10 to 1.50 Pa. Two days after stimulation by shear stress, neurite outgrowth and its direction were measured by F-actin staining and digital image processing. When a shear stress of 0.50 Pa was applied, neurons were most highly aligned with microfibers. The average length of neurite outgrowth with microfibers was largest at a shear stress of 0.25 Pa. The results suggest that micropatterned fibers and fluid-induced shear stress are promising for stimulating neurite outgrowth in a desired direction.

Electrospun PLGA nanofiber scaffolds for articular cartilage reconstruction: mechanical stability, degradation and cellular responses under mechanical stimulation in vitro.

We investigated the potential of a nanofiber-based poly(DL-lactide-co-glycolide) (PLGA) scaffold to be used for cartilage reconstruction. The mechanical properties of the nanofiber scaffold, degradation of the scaffold and cellular responses to the scaffold under mechanical stimulation were studied. Three different types of scaffold (lactic acid/glycolic acid content ratio = 75 : 25, 50 : 50, or a blend of 75 : 25 and 50 : 50) were tested. The tensile modulus, ultimate tensile stress and corresponding strain of the scaffolds were similar to those of skin and were slightly lower than those of human cartilage. This suggested that the nanofiber scaffold was sufficiently mechanically stable to withstand implantation and to support regenerated cartilage. The 50 : 50 PLGA scaffold was degraded faster than 75 : 25 PLGA, probably due to the higher hydrophilic glycolic acid content in the former. The nanofiber scaffold was degraded faster than a block-type scaffold that had a similar molecular weight. Therefore, degradation of the scaffold depended on the lactic acid/glycolic acid content ratio and might be controlled by mixing ratio of blend PLGA. Cellular responses were evaluated by examining toxicity, cell proliferation and extracellular matrix (ECM) formation using freshly isolated chondrocytes from porcine articular cartilage. The scaffolds were non-toxic, and cell proliferation and ECM formation in nanofiber scaffolds were superior to those in membrane-type scaffolds. Intermittent hydrostatic pressure applied to cell-seeded nanofiber scaffolds increased chondrocyte proliferation and ECM formation. In conclusion, our nanofiber-based PLGA scaffold has the potential to be used for cartilage reconstruction.

An in vitro animal study of the biomechanical responses of anulus fibrosus with aging.

STUDY DESIGN: In vitro biomechanical tests were performed on aged (group A) and young (group B) porcine intervertebral discs. OBJECTIVES: The in vitro biomechanical responses of aged and young porcine intervertebral discs were measured under designated axial compressive loads and analyzed. SUMMARY OF BACKGROUND DATA: From the biomechanical point of view, the major biomechanical functions of intervertebral discs are to absorb and distribute external loadings. Although the histological observation of intervertebral discs on the effect of aging and related degeneration has been extensively studied and described, the changes in those biomechanical functions attributable to aging are still left to be studied. METHODS: Two groups were set for mechanical tests. Group A consisted of 24 motion segments obtained from female porcine lumbar (44.0 +/- 2.8-months old). The group B consisted of 30 motion segments from female porcine lumbar (6.2 +/- 1.3 months old). The specimens were chosen randomly from all levels. For the measurements of biomechanical responses, a pressure transducer was placed on anterior and posterolateral locations of anulus fibrosus. Morphological and histological observations were carried out to confirm any age-related changes in both groups. Intradiscal pressures and relaxation times were measured and calculated at points in the anulus fibrosus under designated axial compressive loads. RESULTS: Morphological and histological difference between group A and group B were confirmed with H&E staining and other measurements. Group A showed a lower ratio of nucleus pulposus area to total disc area than did group B. There was no significant difference in the intradiscal pressure between groups as measured in the anterior zone, except at an axial load of 740 N. However, a significant pressure difference was found in the posterolateral zone when the axial load was 542 N or greater (P < 0.05). At 740 N, the average relaxation time for group A was significantly longer than that for group B (P < 0.05). CONCLUSIONS: Differences in biomechanical responses between groups were confirmed. Group A was less flexible and slower at energy relaxation under axial loading. A larger proportion of the external load was taken by the posterolateral part of the degenerative discs. These results were consistent with clinical experiences: 1) most hernias are observed more often at the posterolateral side than other sides, and 2) the degeneration attributable to age reduces the function of absorbing and distribution of external loadings.