Chondrocyte Aggregates Research Articles

Viscoelasticity microenvironment constructed by self-crosslinking hyaluronan hybrid hydrogels regulates chondrogenic differentiation of mesenchymal stem cells

Chondrogenic differentiation of stem cells can be regulated by mechanical properties of biomaterials. Here, hybrid hydrogels, composed of sulfhydrylated hyaluronan (HA-SH) with different molecular weight and collagen I (Col Ⅰ), were designed to investigate chondrogenic differentiation of rabbit bone marrow mesenchymal stem cells (rBMSCs). The enhanced molecular weight of HA-SH decreased the mechanical properties of hybrid hydrogels, but improved viscosity and degradation resistance, which could modulate cell spreading and morphological changes, thereby influencing chondrogenic differentiation. Furthermore, 1 MDa HA-SH combined with Col Ⅰ (1M-HAC) with adequate viscoelasticity was beneficial to the circularity and nuclear chromatin condensation of rBMSCs, which induced significant chondrocyte aggregation, increased glycosaminoglycans and collagen Ⅱ stainings, and reduced collagen Ⅹ staining. This was conducive to preserve a hyaline cartilage's phenotype and reducing inflammation and degeneration. Subcutaneous implantation studies revealed that 1M-HAC also facilitated specific matrix secretion, chondrogenic gene expression and potentially anti-inflammatory and anti-degenerative effects.

The Localizations of Osteoarthritis in the Knee, Ankle and Foot Joints of Cadaver: Comparison in Radiological, Morphological and Histopathological Aspects

Objective: Osteoarthritis (OA) is the most common joint disease. In this study it was aimed to compare the general features of OA such as location, placement, severity and shape of the lesions in terms of radiological and morphological aspects and to determine their relationship with each other. Methods: In our study, the antero-posterior and lateral radiographies of knee talocrural and transverse tarsal joints of 20 cadavers by age between 30 and 50 years were taken. The results obtained from the radiological examination were graded according to the Kellgren and Lawrence scale. For each of the identified regions, the presence of degenerative changes was noted. Then samples were taken from these regions were examined by microscopic methods. The cartilage degeneration changes, presence of fibrillations, density, depth, chondrocyte aggregation, and necrotic changes were evaluated. Results: In the radiological examination OA was found in 35% in knee joint, 25% in the talocrural joint, 15% in the transverse tarsal joint. In the morphological examination OA was found in 31.5% knee joint, 25% ankle joint and 5% transverse tarsal joint. In the microscopic examination OA was found in 94.7% knee joint, in 94.7% ankle joint and in 100% transverse tarsal joint. Conclusion: Although radiological and macroscopic OA was detected in approximately 1/3 of cadavers aged between 30 and 50 years, degeneration of varying degrees was detected in all joints examined in microscopic examination. This shows that an advanced age disease OA, starts at a very early age.

Co-aggregation of MSC/chondrocyte in a dynamic 3D culture elevates the therapeutic effect of secreted extracellular vesicles on osteoarthritis in a rat model

Extracellular vesicles (EVs) have therapeutic effects on osteoarthritis (OA). Some recent strategies could elevate EV's therapeutic properties including cell aggregation, co-culture, and 3D culture. It seems that a combination of these strategies could augment EV production and therapeutic potential. The current study aims to evaluate the quantity of EV yield and the therapeutic effect of EVs harvested from rabbit mesenchymal stem cells (MSCs) aggregates, chondrocyte aggregates, and their co-aggregates in a dynamic 3D culture in a rat osteoarthritis model. MSC and chondrocytes were aggregated and co-aggregated by spinner flasks, and their conditioned medium was collected. EVs were isolated by size exclusion chromatography and characterized in terms of size, morphology and surface markers. The chondrogenic potential of the MSC-ag, Cho-ag and Co-ag EVs on MSC micromass differentiation in chondrogenic media were assessed by qRT-PCR, histological and immunohistochemical analysis. 50 μg of MSC-ag-EVs, Cho-ag-EVs and Co-ag-EVs was injected intra-articularly per knee of OA models established by monoiodoacetate in rats. After 8 weeks follow up, the knee joints were harvested and analyzed by radiographic, histological and immunohistochemical features. MSC/chondrocyte co-aggregation in comparison to MSC or chondrocyte aggregation could increase EV yield during dynamic 3D culture by spinner flasks. Although MSC-ag-, Cho-ag- and Co-ag-derived EVs could induce chondrogenesis similar to transforming growth factor-beta during in vitro study, Co-ag-EV could more effectively prevent OA progression than MSC-ag- and Cho-ag-EVs. Our study demonstrated that EVs harvested from the co-aggregation of MSCs and chondrocytes could be considered as a new therapeutic potential for OA treatment.

Sulfated lactose-modified chitosan. A novel synthetic glycosaminoglycan-like polysaccharide inducing chondrocyte aggregation

Lactose-modified chitosan (CTL) is sulfated using SO3·py or SO3·DMF as sulfating agents. The two products are characterized by elemental analysis, FT-IR, 1H,13C-DEPT-HSQC and 1H,13C-HSQC-TOCSY experiments which allow the extent and selectivity of chemical sulfation to be determined. Dynamic Light Scattering shows a pH-dependent association of the sulfated polysaccharides which are described as flexible by the Smidsrød's B parameter and the intrinsic viscosity at infinite ionic strength. Shear viscosity and intrinsic viscosity show that sulfation protocols lead to chain scission which is more pronounced when SO3·DMF is used. The sulfated samples are able to induce aggregation of human bone marrow mesenchymal stem cells, resulting in the formation of smaller nodules compared to the unmodified CTL sample. Over time, the sample with the higher degree of sulfation allows further aggregation between cell clusters while the sample with the lower degree of sulfation shows dissolution of the aggregates.

Extracellular vesicles derived from starving BMSCs enhance survival of chondrocyte aggregates in grafts by attenuating chondrocyte apoptosis and enabling stable cartilage regeneration for craniofacial reconstruction

The improvement of cell survival in cartilage tissue engineering remains a challenge, especially for large-sized, specifically shaped cartilage grafts used in reconstructing craniofacial defects. In this study, we found that bone marrow mesenchymal stem cells (BMSCs) pre-conditioned in a starving environment enhanced the anti-apoptosis potential of co-transplanted chondrocytes, which significantly enhanced their survival rates before host nutrition was resumed. Further examination revealed that extracellular vesicles (EVs) derived from starving BMSCs played essential roles in ameliorating apoptosis and regulating autophagy of chondrocytes, thereby enhancing the survival of cultured chondrocytes. In vivo studies demonstrated that EVs derived from starving BMSCs significantly improved the survival of chondrocyte bricks, which confirmed the effects of nasal augmentation. These pre-treated chondrocyte bricks showed continuous cartilage growth in vivo and acquired chondrogenesis comparable to that following the chondrocyte-BMSC co-transplantation approach. This study provided new insights on how BMSC-derived EVs improved cartilage reconstruction in the craniofacial regions and offered a new approach for regenerating cartilaginous organs based on cell macroaggregates. Statement of significanceThe use of extracellular vesicles (EVs) of mesenchymal stem cells has been considered as a promising approach in cartilage tissue engineering. In the present study, for the first time, we investigated the protective effect of EVs secreted by starving bone marrow mesenchymal stem cells (BMSCs) on chondrocytes in vitro and in vivo. The results demonstrated that EVs secreted by starving BMSCs inhibited chondrocyte apoptosis and chondrocyte autophagy through many microRNAs, thereby improving the survival of grafts. Transcriptomic analysis revealed the potential mechanisms of this protective effect.

Synovial Mesenchymal Stem Cell-Derived EV-Packaged miR-31 Downregulates Histone Demethylase KDM2A to Prevent Knee Osteoarthritis

Extracellular vesicles (EVs) derived from mesenchymal stem cells (MSCs) have emerged as important mediators of intercellular communication in response to cartilage damage. In this study, we sought to characterize the inhibitory role of microRNA (miR)-31 encapsulated in synovial MSC (SMSC)-derived EVs in knee osteoarthritis (OA). The expression of miR-31, lysine demethylase 2A (KDM2A), E2F transcription factor 1 (E2F1), and pituitary tumor transforming gene 1 (PTTG1) was validated in cartilage tissues of knee OA patients. Following SMSC-EV extraction and identification, chondrocytes with the miR-31 inhibitor were added with SMSC-EVs, whereupon the effects of miR-31 on proliferation and migration of chondrocytes were assessed. The interaction among miR-31, KDM2A, E2F1, and PTTG1 in chondrocyte activities was probed in vitro, along with an in vivo mouse knee OA model. We identified downregulated miR-31, E2F1, and PTTG1 and upregulated KDM2A in cartilage tissues of knee OA patients. SMSC-EV-packaged miR-31 potentiated chondrocyte proliferation and migration as well as cartilage formation by targeting KDM2A. Mechanistically, KDM2A bound to the transcription factor E2F1 and inhibited its transcriptional activity. Enrichment of E2F1 in the PTTG1 promoter region activated PTTG1 transcription, accelerating chondrocyte proliferation and migration. SMSC-EVs and EVs from miR-31-overexpressed SMSCs alleviated cartilage damage and inflammation in knee joints in vivo. SMSC-EV-encapsulated miR-31 ameliorates knee OA via the KDM2A/E2F1/PTTG1 axis.

Increased Expression of Sox9 during Balance of BMSCs/Chondrocyte Bricks in Platelet-Rich Plasma Promotes Construction of a Stable 3-D Chondrogenesis Microenvironment for BMSCs.

Sox9 is an intrinsic transcription factor related to the determination and maintenance of chondrogenic lineage of bone marrow mesenchymal stem cells (BMSCs). In recent research, we have proved that fragmented chondrocyte aggregates (cell bricks) could promote chondrogenesis of BMSCs in vivo. However, it is still unknown whether the ratio of BMSCs/chondrocyte bricks has a significant influence on 3-D cartilage regeneration and related molecular mechanism. To address this issue, the current study subcutaneously injected three groups of cell complex with different rabbit BMSCs/chondrocyte bricks' ratios (1 : 2, 1 : 1, and 2 : 1) into nude mice. Gross morphology observation, histological and immunohistochemical assays, biochemical analysis, gene expression analysis, and western blot were used to compare the influence of different BMSCs/chondrocyte bricks' ratios on the properties of tissue-engineered cartilage and explore the related molecular mechanism. The constructs of 1 : 1 BMSCs/chondrocyte bricks, (B1CB1) group resulted in persistent chondrogenesis with appropriate morphology and adequate central nutritional perfusion without ossification. The related mechanism is that increased expression of Sox9 in the B1C1 group promoted chondrogenesis and inhibited the osteogenesis of BMSCs through upregulating Col-II as well as downregulating RUNX2 and downstream of Col-X and Col-I by upregulating Nkx3.2. This study demonstrated that BMSCs/chondrocyte bricks 1:1 should be a suitable ratio and the Sox9-Nkx3.2-RUNX2 pathway was a related mechanism which played an important role in the niche for stable chondrogenesis of BMSCs constructed by chondrocyte bricks and PRP.

P5. Experimental study on the expression of growth factors in the vertebral growth plate and pedicle in severe idiopathic scoliosis

BACKGROUND CONTEXT Some scoliosis can progress to severe scoliosis with significant wedge deformities in the apex vertebra. Vertebral growth plate is closely related to the longitudinal growth of vertebral body, and the role of the growth factors expression in growth plate in the occurrence and development of severe spinal deformities has not been studied so far. PURPOSE To investigated the expression of multiple growth factors in the concave and convex vertebral growth plate and pedicle in severe idiopathic scoliosis. STUDY DESIGN/SETTING A PATIENT SAMPLE Severe scoliosis and no significant cardiopulmonary dysfunction, no significant malnutrition, and no abnormal bone metabolism were included in this study. Congenital, neuromuscular, neurofibromatosis, connective tissue disease and syndrome scoliosis were excluded. OUTCOME MEASURES The specimens were observed histologically. Immunohistochemistry was used to observe the differences of TGF, FGF, BMP and IGF expression. METHODS Severe scoliosis and no significant cardiopulmonary dysfunction, no significant malnutrition, and no abnormal bone metabolism were included in this study. Congenital, neuromuscular, neurofibromatosis, connective tissue disease and syndrome scoliosis were excluded. Growth plate and pedicle specimens of convex and concave side of the vertebra in the apical region were obtained during the PVCR. The specimens were observed histologically. Immunohistochemistry was used to observe the differences of TGF, FGF, BMP and IGF expression in the pedicle and growth plate of concave and convex side of the apex vertebra. Semi-quantitative analysis was carried out by immunohistochemical image analysis system. Statistical analysis was carried out on the results, with P RESULTS Ten patients (6 male, 4 female) were included in this study, with an average age of 17.6 years, and the apex vertebra was located between T6 to T12. The preoperative mean coronal deformity was 127.7° (96°-158°), and the sagittal deformity was 115° (85°-150°).The cells in the cartilage layer of the apex vertebral were disordered, the cartilage matrix was not uniform, and the concave side was thin. The fibrosis was aggravated in the matrix and accompanied by focal calcification. Some chondrocyte aggregation was observed. Bone density increased in the concave side of the pedicle. Immunohistochemical staining results showed that FGF, BMP and TGF had basically similar expression characteristics, and the expressions in the convex side of growth plate and pedicle of the apex were higher than the concave side, which had statistical significance(p CONCLUSIONS The expressions of growth factors in the growth plate and pedicle of the apex were different in patients with severe idiopathic scoliosis, and the imbalance of growth factor expression is one of the factors that cause significant deformity in such patients. Abnormal expression may lead to the significant development of the deformity, and the abnormal mechanical environment caused by the significant deformity in the later stage may jointly lead to the further development of the deformity. FDA DEVICE/DRUG STATUS This abstract does not discuss or include any applicable devices or drugs.

Dynamic formation of cellular aggregates of chondrocytes and mesenchymal stem cells in spinner flask.

ObjectivesCellular aggregates are readily applicable in cell‐based therapy. The effects of agitation and inoculation density on the aggregation of cells in spinner flask and the molecular mechanism of aggregation were investigated.Materials and methodsThe aggregation kinetics of cells in spinner flask was evaluated with bovine articular chondrocytes (bACs), rabbit bone marrow‐derived mesenchymal stem cells (rMSCs) and their mixture. The morphology of cellular aggregates was studied with scanning electron microscopy and gene expression of cell adhesion‐related molecules was analysed.ResultsIt was shown that suspension culture in spinner flask induced the aggregation of bACs and rMSCs. Both cells exhibited increased aggregation rate and aggregate size with decreasing agitation rate and increasing cell inoculation density. Additionally, aggregate size increased with extended culture time. By analysing gene expression of integrin β1 and cadherin, it was indicated that these molecules were potentially involved in the aggregation process of bACs and rMSCs, respectively. Aggregates composed of both bACs and rMSCs were also prepared, showing rMSCs in the core and bACs in the periphery.ConclusionsCellular aggregates were prepared in dynamic suspension culture using spinner flask, the key parameters to the aggregation process were identified, and the molecular mechanism of aggregation was revealed. This would lay a solid foundation for the large‐scale production of cellular aggregates for cell‐based therapy, such as cartilage regeneration.

Effectiveness of exercise-induced cytokines in alleviating arthritis symptoms in arthritis model mice.

Recently, health awareness in Japan has been increasing and active exercise is now recommended to prevent lifestyle-related diseases. Cytokine activities have many positive effects in maintaining the health of a number of organs in the body. Myokines are cytokines secreted by skeletal muscles in response to exercise stimulation, and have recently generated much attention. Around 700,000 patients in Japan suffer from rheumatoid arthritis, making it the most prevalent autoimmune disease that requires active prevention and treatment. In the present study, a mouse model of spontaneous arthritis (SKG/Jcl) was subjected to continuous exercise stimulation, starting before the disease onset, to examine the effects of anti-inflammatory and inflammatory cytokine secretion on arthritis. For this stimulation, we developed a device that combines shaking and vibration. The results revealed that exercise stimulation delayed the onset of arthritis and slowed its progression. Thickened articular cartilage and multiple aggregates of chondrocytes were also observed. Further, exercise stimulation increased the expression of IL-6, IL-10, and IL-15, and inhibited TNF-α expression. From these results, we infer that the anti-inflammatory effects of IL-6 and IL-10, which showed increased expression upon exercise stimulation, inhibited the inflammatory activity of TNF-α and possibly delayed the onset of arthritis and slowed its progression. Novel methods for preventing and treating arthritis under clinical settings can be developed on the basis of these findings.

Synoviocyte Derived-Extracellular Matrix Enhances Human Articular Chondrocyte Proliferation and Maintains Re-Differentiation Capacity at Both Low and Atmospheric Oxygen Tensions.

BackgroundCurrent tissue engineering methods are insufficient for total joint resurfacing, and chondrocytes undergo de-differentiation when expanded on tissue culture plastic. De-differentiated chondrocytes show poor re-differentiation in culture, giving reduced glycosaminoglycan (GAG) and collagen matrix accumulation. To address this, porcine synoviocyte-derived extracellular matrix and low (5%) oxygen tension were assessed for their ability to enhance human articular chondrocyte expansion and maintain re-differentiation potential.MethodsPorcine synoviocyte matrices were devitalized using 3 non-detergent methods. These devitalized synoviocyte matrices were compared against tissue culture plastic for their ability to support human chondrocyte expansion. Expansion was further compared at both low (5%), and atmospheric (20%) oxygen tension on all surfaces. Expanded cells then underwent chondrogenic re-differentiation in aggregate culture at both low and atmospheric oxygen tension. Aggregates were assessed for their GAG and collagen content both biochemically and histologically.ResultsHuman chondrocytes expanded twice as fast on devitalized synoviocyte matrix vs. tissue culture plastic, and cells retained their re-differentiation capacity for twice the number of population doublings. There was no significant difference in growth rate between low and atmospheric oxygen tension. There was significantly less collagen type I, collagen type II, aggrecan and more MMP13 expression in cells expanded on synoviocyte matrix vs. tissue culture plastic. There were also significant effects due to oxygen tension on gene expression, wherein there was greater collagen type I, collagen type II, SOX9 and less MMP13 expression on tissue culture plastic compared to synoviocyte matrix. There was a significant increase in GAG, but not collagen, accumulation in chondrocyte aggregates re-differentiated at low oxygen tension over that achieved in atmospheric oxygen conditions.ConclusionsSynoviocyte-derived matrix supports enhanced expansion of human chondrocytes such that the chondrocytes are maintained in a state from which they can re-differentiate into a cartilage phenotype after significantly more population doublings. Also, low oxygen tension supports GAG, but not collagen, accumulation. These findings are a step towards the production of a more functional, tissue engineered cartilage.

Acute Left-sided Foot Drop Attributed to Recurrent Synovial Chondromatosis of the Lumbar Spine

Objective: To discuss an uncommon case of nerve root compression caused by synovial chondromatosis of the lumbar spine. Summary of background: Synovial chondromatosis is an uncommon, benign monoarthritic condition of the synovial lining of large joints of the body. It is considered a metaplastic process rather than a neoplastic one, in which nodules are formed from aggregates of chondrocytes in the synovium. The nodules cause non-specific symptoms such as pain and decreased range of motion in the affected joint. In rare instances, synovial chondromatosis has been known to involve the spine, with associated radiculopathy. We report a case of synovial chondromatosis of the lumbar spine, which to our knowledge is the first case with foot drop. Case presentation: A 58-year-old woman presented with symptoms of lower back pain with unilateral radiation, numbness, and tingling. After primary resection of an extradural cartilaginous mass in the region of L4-L5, her symptoms remitted. One year post-operatively, she experienced a progressive recurrence of her symptoms and the acute onset of foot drop. Re-excision provided partial symptomatic relief. Histopathology showed nodular aggregates of benign cartilage, consistent with synovial chondromatosis. Conclusion: Synovial chondromatosis of the spine is a rare but potentially challenging problem for both patients and clinicians. Due to the broad clinical presentation and vague imaging profile, preoperative diagnosis is difficult. The histopathologic findings must be correlated clinically to arrive at the correct diagnosis. Surgical removal may resolve the symptoms, though recurrence requiring re-excision is not uncommon. This is the fourth reported case of synovial chondromatosis involving the lumbar spine, and the first with recurrence resulting in an exacerbation of prior symptoms including foot drop.

715 プロリン含有周期性ペプチドを用いた軟骨細胞凝集体の機能評価(GS-2 生体材料)

715 プロリン含有周期性ペプチドを用いた軟骨細胞凝集体の機能評価(GS-2 生体材料)

Observation and Quantification of Chondrocyte Aggregation Behavior on Fibroin Surfaces Using Voronoi Partition

Cell migration is one of the fundamental processes in histogenesis, and it is necessary to investigate such multicellular behavior quantitatively in cell regeneration studies. In this study, Voronoi diagram analysis was first confirmed in simulation testing, and then used to evaluate the multicellular behavior of chondrocytes on three different substrates: (1) wild-type fibroin (FIB); (2) L-RGDSx2 transgenic fibroin; (3) and collagen. The indices for the round factor average, round factor homogeneity, and area disorder (AD), calculated from Voronoi diagram analysis, were used to characterize the difference in spatiotemporal changes for the different chondrocyte populations, and a regression analysis of the AD index was used to measure the speed of cell aggregation. The results suggested that the arginine-glycine-aspartic acid-serine sequence affects aggregate formation of chondrocytes cultured on FIB. The Voronoi diagram analysis represents one of the promising quantitative analyses for cell regeneration studies.

Modulation of chondrocyte migration and aggregation by insulin-like growth factor-1 in cultured cartilage

The effect of insulin-like growth factor-1 (IGF-1) on the behavior of rabbit chondrocytes in cultured collagen (CL) gels initially seeded with 2×10(5) cells/ml was examined. On day 5, the frequency of migrating cells cultured in presence of 100ng IGF-1/ml was 0.04, which was 54% of the frequency in IGF-1-free culture. The presence of IGF-1 caused an increase in the frequency of dividing cells from 0.09 to 0.13. These results suggest that IGF-1 suppressed the migration of chondrocytes in the CL gels while stimulating cell division in the initial culture phase. The proteolytic migration of cells was thought to be suppressed by the down-regulation of membrane type 1 matrix metalloproteinase by IGF-1. This contributed to the formation of aggregates with spherical-shaped cells that produced collagen type II.

J028011 細胞間接着が軟骨組織形成に及ぼす影響

We reported that we could treat the defect of patella by using fibroin which is from silkworm cocoon. And we also reported that we observed the rich cartilage matrixes and chondrocyte's aggregation in fibroin. So we focused on the chondrocyte's aggregation on fibroin and tried to develop the method of analyzing it quantitatively for evaluating the effect of cell-cell interaction on aggregated chondrocyte cultured on fibroin or other matrixes. First, we tried to define cell aggregation by comparing chondrocyte's aggregation on fibroin with random walk models. And second, we cultured chondrocytes on fibroin, fibronectin and collagen type I and on we evaluated the amount of GAG production and the number of chondrocytes cultured on each matrixes. As a result, at first we found that on fibroin the number of cells which are 10-20 |0.m away from other cells is especially high. And also, they keep cell-cell adhesion for more than an hour. And second, it is suggested that the chondrocyte on fibroin is easier to aggregate than that on fibronectin or collagen type I. What is more, the chondrocyte on fibroin has a higher ability to produce GAG than that on other matrixes. From these results it is suggested that the aggregation movement on fibroin is caused by cell-cell adhesion but also such cell-cell interaction is affected by cell-matrix interaction. In addition it is also suggested that the aggregation movement is related to the ability of chondrogenesis.

B204 異なる培養環境が軟骨細胞の凝集運動に与える影響(B2-1 軟骨のバイオメカニクス)

B204 異なる培養環境が軟骨細胞の凝集運動に与える影響(B2-1 軟骨のバイオメカニクス)

Spinner-flask culture induces redifferentiation of de-differentiated chondrocytes

Implantation of chondrocytes isolated from patients and expanded in number in vitro is being used to treat patients with cartilage injuries. However, chondrocytes de-differentiate during culture with several passages, and cartilage regenerated by implantation of de-differentiated chondrocytes may be suboptimal. Here, we show that a spinner-flask culture system induces formation of chondrocyte aggregates and redifferentiate de-differentiated chondrocytes. Spinner-flask cultures induced the aggregate formation of chondrocytes with passage 1 or 4. Importantly, spinner-flask cultures induced redifferentiation of the de-differentiated chondrocytes, as type I collagen expression was significantly lower and type II collagen expression was significantly higher in spinner flask-cultured chondrocytes than in monolayer-cultured chondrocytes. This system is easily scalable and could be feasible for clinical setting.

Konjac glucomannan-based hydrogel with hyaluronic acid as a candidate for a novel scaffold for chondrocyte culture

Chondrocytes were cultured using konjac glucomannan (KGM) and hyaluronic acid (HA) as a scaffold for cartilage regeneration. They were subsequently compared with scaffolds produced using agarose hydrogels. Chondrocytes derived from Japanese white rabbits were cultured: 2.0 x 10(5) cells were seeded on KGM containing hyaluronic acid (KGM/HA) and agarose and cultured for 5 days. Their viability was assayed using WST-8 procedures; the ultimate stress and modulus of elasticity of each construct was calculated. After 3 days of cultivation, mRNA in chondrocytes, such as collagen types I and II and aggrecan, were measured using RT-PCR. Both chondrocyte-seeded constructs were stained with safranin O/fast green and were evaluated histologically. Chondrocyte viability decreased concomitantly with increasing KGM/HA or agarose concentration and with culture time. Cell viability in 2% agarose was significantly lower than that in 2% KGM/HA on the third and fifth days (p < 0.05). The primary elastic modulus increased concomitantly with increasing polysaccharide concentration. Elastic moduli of 2% KGM/HA with chondrocytes (0.389 +/- 0.119 N/mm(2)) showed little difference from those without chondrocytes (0.283 +/- 0.243 N/mm(2)), although those of 2% agarose with chondrocytes (0.403 +/- 0.094 N/mm(2)) were significantly lower than those without chondrocytes (0.736 +/- 0.227 N/mm(2); p < 0.05). Collagen type II mRNA expression was higher in KGM/HA and agarose than in monolayer cultures, although KGM/HA had lower aggrecan mRNA expression levels than did agarose. Histological tests of KGM/HA-chondrocyte constructs revealed chondrocyte aggregation and proteoglycan production in the pericellular region. The results show that KGM/HA might be useful for chondrocyte culture.

A collagen-coated surface enables quantitative evaluation of morphological behaviors of rabbit chondrocytes relating to cell differentiation in an early culture phase

A surface with collagen type I coating was used as a tool mimicking three dimensional environment for studying the behaviors of rabbit chondrocyte aggregates found in an early culture phase. The morphology of cell aggregates was evaluated quantitatively in terms of a morphology-relating parameter of specific branching factor, Z s, determined under varied culture conditions. Branching in aggregates increased along with proceeding of culture from 5 to 6 days when seeded at a lower density of X 0 = 1.0 × 10 3 cells/cm 2, giving Z s = 0.24 tips/cell at 6 days, the value of which was 1.7 times than that obtained from the culture with a higher seeding density of X 0 = 1.0 × 10 4 cells/cm 2 ( Z s = 0.14 tips/cell). This value significantly reduced to Z s = 0.13 tips/cell in the culture with X 0 = 1.0 × 10 3 cells/cm 2 by using the conditioned medium prepared from the foregone culture with X 0 = 1.0 × 10 4 cells/cm 2, inducing the morphological architecture and collagen type II production of aggregated cells with the similarities to those in the standard culture seeded at 1.0 × 10 4 cells/cm 2. The mRNA expressions of differentiation-marker genes (collagen types I and II) were confirmed to be regulated in accordance with the morphological analyses as well as the extracellular matrix formation of aggregates in the both cultures conducted with the standard and conditioned media. The present study suggested that soluble factor(s) secreted from the chondrocytes at the higher seeding density modulates the cell aggregation and the state of chondrogenic differentiation through encouraging cell–cell communications.