Ligament Cells Research Articles

In Vitro and In Vivo Behaviors of the Three-layered Nanocarbonated Hydroxyapatite/Collagen/PLGA Composite

A three-layered nano-carbonated hydroxyapatite/collagen/poly (lactic-co-glycolic acid) (nCHAC/PLGA) composite membrane that consisted of 8 wt% nCHAC + PLGA/4 wt%nCHAC + PLGA/PLGA was developed using a layer-by-layer casting. The human periodontal ligament and osteoblastic MC3T3-E1 cells were cultured on the three-layered composite membrane; the co-culture gave a more positive response than the pure PLGA membrane. Subsequently, the composite and pure PLGA membranes were implanted into critical 6.2 mm defects in rat crania. After 4 weeks, significant healing was observed in the defects implanted with the nCHAC/PLGA composite membrane, while similar healing was only observed with pure PLGA implants after 8 weeks. This novel composite appears to be a very promising candidate for periodontal and bone defects therapy.

Lentiviral-mediated correction of MPS VI cells and gene transfer to joint tissues

Joint disease in mucopolysaccharidosis type VI (MPS VI) remains difficult to treat despite the success of enzyme replacement therapy in treating other symptoms. In this study, the efficacy of a lentiviral vector to transduce joint tissues and express N-acetylgalactosamine-4-sulphatase (4S), the enzyme deficient in MPS VI, was evaluated in vitro and the expression of β-galactosidase was used to evaluate transduction in vivo. High viral copy number was achieved in MPS VI fibroblasts and 4-sulphatase activity reached 12 times the normal level. Storage of accumulated glycosaminoglycan was reduced in a dose dependent manner in both MPS VI skin fibroblasts and chondrocytes. Enzyme expression was maintained in skin fibroblasts for up to 41 days. Comparison of two promoters; the murine phosphoglycerate kinase gene promoter (pgk) and the myeloproliferative sarcoma virus long terminal repeat promoter (mpsv), demonstrated a higher level of marker gene expression driven by the mpsv promoter in both chondrocytes and synoviocytes in vitro. When injected into the rat knee, the expression of β-galactosidase from the mpsv promoter was widespread across the synovial membrane and the fascia covering the cruciate ligaments and meniscus. No transduction of chondrocytes or ligament cells was observed. Transduction was maintained for at least 8 weeks after injection. These results indicate that the lentiviral vector can be used to deliver 4S to a range of joint tissues in vitro and efficiently transduce synovial cells and express β-galactosidase in vivo.

Expression of alpha, beta and gamma subunits of epithelial sodium channel in the cochlea and endolymphatic sac of guinea pig

To investigate the distribution and role of alpha, beta and gamma subunits of epithelial sodium channel (ENaC) in the cochlea and endolymphatic sac of guinea pig. The expression of alpha-, beta- and gamma-ENaC subunits proteins was studied by immunohistochemistry with the specific polyclonal rabbit antibodies against the alpha, beta and gamma subunits of rat ENaC. Alpha-ENaC mRNA was detected by in situ hybridization with digoxin labeled cDNA probe. All three subunits of ENaC, alpha-, beta- and gamma-, were widely distributed in the labyrinth. In the cochlea, strong labeling of alpha-ENaC protein was found in the spiral limbus, and to a less extent, in the spiral ligament, organ of Corti and Reissner's membrane. The immunoreactivity of beta-ENaC was observed in the spiral ligament, spiral limbus, spiral ganglion, organ of Corti and Reissner's membrane with a less intensity than that of alpha-ENaC. Gamma-ENaC was presented primarily in the superior part of the spiral ligament, spiral limbus, spiral ganglion, and weakly in the organ of Corti and Reissner's membrane. In the endolymphatic sac, intensive immunoreactivities of all three subunits were seen in the epithelial cells and the subepithelial cells at similar intensity. Alpha-ENaC mRNA was localized in the spiral limbus, the inferior part of spiral ligament, stria vascularis, and epithelial cells and subepithelial cells of endolymphatic sac. Different subunits of the ENaC expressed in various cell regions of the cochlea and endolymphatic sac in distinct patterns may form the functional sodium channel to regulate the endolymph, thus serve to maintain homeostasis in inner ear.

Tissue specific characteristics of cells isolated from human and rat tendons and ligaments

BackgroundTendon and ligament injuries are common and costly in terms of surgery and rehabilitation. This might be improved by using tissue engineered constructs to accelerate the repair process; a method used successfully for skin wound healing and cartilage repair. Progress in this field has however been limited; possibly due to an over-simplistic choice of donor cell. For tissue engineering purposes it is often assumed that all tendon and ligament cells are similar despite their differing roles and biomechanics. To clarify this, we have characterised cells from various tendons and ligaments of human and rat origin in terms of proliferation, response to dexamethasone and cell surface marker expression.MethodsCells isolated from tendons by collagenase digestion were plated out in DMEM containing 10% fetal calf serum, penicillin/streptomycin and ultraglutamine. Cell number and collagen accumulation were by determined methylene blue and Sirius red staining respectively. Expression of cell surface markers was established by flow cytometry.ResultsIn the CFU-f assay, human PT-derived cells produced more and bigger colonies suggesting the presence of more progenitor cells with a higher proliferative capacity. Dexamethasone had no effect on colony number in ACL or PT cells but 10 nM dexamethasone increased colony size in ACL cultures whereas higher concentrations decreased colony size in both ACL and PT cultures. In secondary subcultures, dexamethasone had no significant effect on PT cultures whereas a stimulation was seen at low concentrations in the ACL cultures and an inhibition at higher concentrations. Collagen accumulation was inhibited with increasing doses in both ACL and PT cultures. This differential response was also seen in rat-derived cells with similar differences being seen between Achilles, Patellar and tail tendon cells. Cell surface marker expression was also source dependent; CD90 was expressed at higher levels by PT cells and in both humans and rats whereas D7fib was expressed at lower levels by PT cells in humans.ConclusionThese data show that tendon & ligament cells from different sources possess intrinsic differences in terms of their growth, dexamethasone responsiveness and cell surface marker expression. This suggests that for tissue engineering purposes the cell source must be carefully considered to maximise their efficacy.

Basic fibroblast growth factor regulates expression of heparan sulfate in human periodontal ligament cells

Heparan sulfate (HS) proteoglycan is a widely distributed biological molecule that mediates a variety of physiological responses in development, cell growth, cell migration, and wound healing. We examined the effects of basic fibroblast growth factor-2 (FGF-2), which is known to modulate extracellular matrix (ECM) production of various cell types, on the production of HS proteoglycan by human periodontal ligament (HPDL) cells. We also examined the effects of FGF-2 on the expression of syndecans, a major family of membrane-bound HS proteoglycans. Treatment of HPDL cells with FGF-2 for 72 h resulted in a pronounced increase in the level of HS in the culture supernatant in a dose-dependent manner. However, reverse transcription-polymerase chain reaction data (RT-PCR) revealed that FGF-2 marginally reduced the gene expression of syndecan-1, -2, and -4, and did not alter the level of syndecan-3 mRNA. Furthermore, FGF-2 did not have an effect on the mRNA expression of enzymes associated with HS biosynthesis. Interestingly, FACS analysis revealed that the syndecan family displayed diverse alterations in response to FGF-2. FGF-2 barely altered the expression of syndecan-1, but decreased the expression of syndecan-2 and -4 on HPDL cells. Moreover, dot blot analysis showed that FGF-2 did not alter the level of syndecan-1 and -2, but enhanced the level of syndecan-4 in culture supernatants of FGF-2-stimulated HPDL cells. These results suggest that the FGF-2-activated increase in the level of HS in conditioned medium may be a result of shedding of syndecan-4 from the HPDL cell surface. Taken together, FGF-2 may differentially regulate the expression of HS proteoglycans in a HS-proteoglycan-subtype-dependent manner. The diversity of the expression patterns of HS proteoglycans may be associated with the FGF-2-induced biological functions of HPDL cells.

Structure and In Vitro Bioactivity of Si Enriched Hydroxyapatite/TiO<sub>2</sub> Coatings on Pure Titanium by Micro-Arc Oxidation

The effect of Si doping on the bioactivity and characteristics of hydroxyapatite/TiO2 composite films was investigated. Pure titanium plates were treated to form Ca, P and Si enriched titania-based films by using micro-arc oxidation (MAO). Then, some specimens were undergone a further hydrothermal treatment to produce a thin outermost layer of hydroxyapatite (HA). Experiments revealed that that the film, which was about 10μm thick, was composed of amorphous silicon dioxide, amorphous calcium phosphate, rutile and anatase. The film was porous and uneven, with the pore size of 1-4μm. No obvious cracks exist on the surface. In order to investigate the cytocompatibility of the films, periodontal ligament (PDL) cells were seeded onto the surface of the films. It was concluded that the Si ion did not influence the characters of MAO film but Si enriched hydroxyapatite/ TiO2 composite coatings exhibited an effect on cell attachment.

Collagen Synthesis in Tenocytes, Ligament Cells and Chondrocytes Exposed to a Combination of Glucosamine HCl and Chondroitin Sulfate

Clinical testing of the nutraceuticals glucosamine (glcN) and chondroitin sulfate (CS) has shown efficacy in providing relief from symptoms in osteoarthritic patients. In vitro and in vivo studies support existence of a synergistic relationship upregulating synthetic activity in chondrocytes. A combination of glcN and CS may also be useful as adjunct therapy in sports-related injuries if similar upregulation of collagen synthesis is elicited in accessory ligament and tendon joint tissue. Collagen and non-collagenous protein (NCP) synthesis in cultures of bovine tenocytes, ligament cells and chondrocytes exposed to glcN + CS were assayed by uptake of radiolabeled proline into collagenase-sensitive material. Assay of radiolabel in hydroxyproline (a specific marker for collagen synthesis) following HPLC isolation confirmed the specificity of the metabolic effect. Synthesis of total collagenase-sensitive material was maximally upregulated at physiologically obtainable doses of glcN + CS. Tissue response followed the sequence ligament cells (+69%) > chondrocytes (+56%) > tenocytes (+22%). Labeled hydroxyproline increased by 132% in ligament cells, 27% in tenocytes and 49% in epitendon cells after a 48 h exposure to 5 μg ml−1 glcN + 4 μg ml−1 CS. Low dose combinations of glcN and CS effectively stimulate in vitro collagen and NCP synthesis by ligament cells, tenocytes and chondrocytes. Hence, therapeutic use following accessory joint tissue trauma may help augment repair processes.

The differentiation of mesenchymal stem cells by mechanical stress or/and co-culture system

Differentiation of mesenchymal stem cells (MSCs) into anterior cruciate ligament (ACL) cells is regulated by many factors. Mechanical stress affects the healing and remodeling process of ACL after surgery in important ways. Besides, co-culture system had also showed the promise to induce MSCs toward different kinds of cells on current research. The purpose of this study was to investigate the gene expression of ACL cells’ major extracellular matrix (ECM) component molecules of MSCs under three induction groups. In addition, to follow our previous study, cell electrophoresis technique and mRNA level gene expression of MSC protein were also used to analyze the differentiation of MSCs. The results reveal that specific regulatory signals which released from ACL cells appear to be responsible for supporting the selective differentiation toward ligament cells in co-culture system and mechanical stress promotes the secretion of key ligament ECM components. Therefore, the combined regulation could assist the development of healing and remolding of ACL tissue engineering. Furthermore, this study also verifies that cell electrophoresis could be used in investigation of cell differentiation. Importantly, analysis of the data suggests the feasibility of utilizing MSCs in clinical applications for repairing or regenerating ACL tissue.

Amelogenin-mediated Regulation of Osteoclastogenesis, and Periodontal Cell Proliferation and Migration

We previously reported that amelogenin isoforms M180 and leucine-rich amelogenin peptide (LRAP) are expressed in the periodontal region, and that their absence is associated with increased cementum defects in amelogenin-knockout (KO) mice. The aim of the present study was to characterize the functions of these isoforms in osteoclastogenesis and in the proliferation and migration of cementoblast/periodontal ligament cells. The co-cultures of wild-type (WT) osteoclast progenitor and KO cementoblast/periodontal ligament cells displayed more tartrate-resistant acid phosphatase (TRAP)-positive cells than the co-cultures of WT cells. The addition of LRAP to both co-cultures significantly reduced RANKL expression and the TRAP-positive cells. Proliferation and migration rates of the KO cementoblast/periodontal ligament cells were lower than those of WT cells and increased with the addition of either LRAP or P172 (a porcine homolog of mouse M180). Thus, we demonstrate the regulation of osteoclastogenesis by LRAP, and the proliferation and migration of cementoblast/periodontal ligament cells by LRAP and P172.

Effects of cigarette smoking on early medial collateral ligament healing in a mouse model

Cigarette smoking delays the healing process and increases morbidity associated with many common musculoskeletal disorders such as medial collateral ligament (MCL) injury. In the current study, a murine model of MCL healing was used to test the hypothesis that smoking impairs extracellular matrix synthesis after injury. Mice were divided into two groups, a nonsmoking control group and a group exposed to smoke for 2 months prior to surgical MCL injury. Mice were euthanized at 3 and 7 days after surgery. Subsequently, propidium iodine staining was used to quantify cellular density of injured and sham ligaments. Immunohistochemical staining and in situ hybridization to mRNA were used to detect proliferation, apoptosis, and type I collagen gene expression at the site of injury. Cell density increased significantly from baseline to 7 days after injury in control mice. In mice exposed to cigarette smoke, there was a significantly lower cellular density compared to controls at this time point (p=0.01). There was no difference in proliferation between groups at the site of injury, and the low level of proliferation observed was not sufficient to account for the large increase in cell density by day 7. No evidence of apoptosis was observed in any of the groups at the site of injury. Type I collagen gene expression was higher in controls compared to smokers at day 7. Almost all of the cells in the substance of the injured MCL at day 7 were spindle-shaped and expressed type I collagen, suggesting that increased cell density from day 3 to day 7 represented an increase in ligament cells rather than an increased inflammatory response. We conclude that the decreased cellular density and type I collagen expression in the injured ligament of mice exposed to smoke begin to provide a cellular and molecular basis for delayed or deficient early healing in these animals.

Cell adhesion of bone marrow cells, chondrocytes, ligament cells and synovial cells on a PLGA–collagen hybrid mesh

Tissue engineering has been rapidly developing as an alternative approach to the repair and regeneration of damaged human tissue. A temporary scaffold is necessary to accommodate new cells and to guide the formation of tissue. Cell adhesion to a scaffold is the first step of cell behavior when it comes in contact with the scaffold surface and is an important consideration in tissue engineering. Bone marrow cells, chondrocytes, ligament cells and synovial cells were cultured in a PLGA–collagen hybrid mesh and their adhesion to the scaffold was studied. All four kinds of cells adhered to the hybrid mesh. The cells did not spread and showed a round morphology after culture for 30 min either in serum or in a serum-free medium. If cells were seeded at a high concentration, more cells were observed in the hybrid mesh. After culture for 6 h, the cells spread and showed a flatter morphology. The cells cultured in the serum medium spread out more than did those in serum-free medium. Calculations of cell seeding efficiencies were based on the number of cells that adhered to the mesh after culture for 30 min and for 6 h. The number of adhered cells increased with an increase of culture time for all four types of cells. For the mesenchymal stem cells, the medium composition and cell concentration affected seeding efficiency during the initial period. Cell seeding efficiency increased with an increase in cell concentration and the serum medium promoted cell adhesion, but not after culture for 6 h. The adhesion of the chondrocytes and ligament cells showed the same trend in that the efficiency of cell growth did not change in serum or serum-free medium, or in high or low cell concentration. For the synovial cells, cell concentration did not affect cell seeding efficiency, but more cells adhered to the hybrid mesh in the serum-free medium than did in the serum medium. These results suggest that the seeding conditions should be specified according the specific cell type when the PLGA–collagen hybrid mesh is used for tissue engineering.

Ability of a bovine bone graft, alone or enriched with PDGF-BB or rhBMP-2, to promote human periodontal ligament (PDL) cells proliferation. A preliminary study.

One of the most important goals of the periodontal therapy procedures is to stimulate the formation of new bone into osseous defects resulted from periodontal disease. A wide range of grafting materials is used to achieve this aim. Recently, the Human Tissue Bank of the National Center for Scientific Research 'Demokritos' in Athens (Greece) has prepared, in a preliminary study, a cancellous bovine-derived bone matrix (BBM). The purpose of the present work was to investigate the role of this bovine bone material in the periodontal regeneration, by studying the rate of human periodontal ligament (PDL) cells proliferation in the presence of this matrix alone, or after the addition of the growth factors, platelet-derived growth factor-BB (PDGF-BB) or recombinant human bone morphogenetic protein-2 (rhBMP-2).Bovine bone graft was prepared using the 'know how' acquired by the 30 years continuous preparation and delivery of lyophilized human bone grafts by the 'Demokritos' Bank.PDL cells cultures were derived from the mid root of two maxillary premolars. The teeth were caries-free and were extracted for orthodontic reasons from 1 adult female patient. Cells were grown in 24-well dishes in the presence of 20 mg BBM. On day 2 of quiescence, new medium was added with 10 ng/ml of PDGF-BB or 50 ng/ml of rhBMP-2. To determine the effects of the test agents on cell proliferation, DNA synthesis was estimated by measuring [(3)H] thymidine incorporation. After 48 h of incubation the cells were processed to subject to scintillation counting. Counts per minute (cpm/well) were determined for each sample.The results revealed that this BBM has the ability to maintain PDL cells proliferation and could be used as an alternative graft material. PDGF-BB when added improved the cell proliferative response resulting in a more active BBM, while the presence of rhBMP-2 did not support cell mitosis.

Development of the attachment zones in the rat anterior cruciate ligament: changes in the distributions of proliferating cells and fibrillar collagens during postnatal growth

The development of the attachment zones of the anterior cruciate ligament (ACL) is an important consideration when examining the structural properties. The aim of this study was to elucidate the morphological changes and the distribution of proliferating cells and collagen types I, II and III at the attachment zones of the rat ACL during postnatal growth. The majority of proliferating cell nuclear antigen (PCNA) immunostained cells were noted near the ligament insertion, especially at the tibial site, and these cells gradually changed to fibrochondrocyte-like cells but still produced collagen types I and III at birth until one month old when rapid longitudinal growth of the ACL took place. After one month when the rate of the ligament growth decreased to one thirtieth of that during the first month and the epiphyseal cartilage at the attachment zone had been replaced by bone, these fibrochondrocyte-like cells began to produce collagen type II and reveal safranin O staining. The immunolabelling pattern to collagen type III was similar to that of PCNA immunostaining during the growth phase. Our findings show that the fibrochondrocytes at the attachment zone may develop from the ligament cells and act as a growth zone for the ligament during the period of ligament growth, and that subsequently, these cells begin to synthesis collagen type II and proteoglycans after epiphyseal ossification. These observations mainly occurred at the tibial attachment zone.

Potential periodontal regeneration by application of monoclonal antibodies against integrin-subunits a6 and b1

Successful guided tissue regeneration (GTR) should result in a functional attachment apparatus
 of the periodontium. The crucial points in the healing process are potential microbiological
 colonization of mechanical barrier membranes, lacking contact to the connective tissue and apical
 growth of the gingival epithelium. The membranes‘ success might be improved by equipping them
 with antibiotics, specific inhibitors for the epithelial growth and growth factors for periodontal
 ligament cells (PLC) or progenitor cells. Monoclonal antibodies (mAb) directed to integrin subunits
 a6 and b1 were tested for their specificity to epithelial growth. In vitro assays were performed as
 direct and indirect contact by seeding the human HaCaT-cell-line and gingival fibroblasts.

Effects of cell adhesion molecules on adhesion of chondrocytes, ligament cells and mesenchymal stem cells

Smooth cell seeding and cell adhesion on temporary porous scaffolds are critical for tissue engineering. We investigated that the effects of type I collagen, type II collagen, fibronectin, vitronectin and poly- l-lysine, laminin, chondroitin-4-sulfate, chondroitin-6-sulfate, aggrecan and hyaluronic acid on adhesion of articular chondrocytes, ligament cells and mesenchymal stem cells (MSCs), for well understanding the interaction between adhesion molecules and diverse kinds of cells. The cell adhesion molecules were coated on the surfaces of 96-well culture plates. The cells were inoculated and cultured in the wells for 30 min, and subsequently, the number of adhered cells was determined. Type I collagen, type II collagen, fibronectin, vitronectin and poly- l-lysine promoted significantly more cell adhesion than laminin, chondroitin-4-sulfate, chondroitin-6-sulfate, aggrecan and hyaluronic acid. MSCs showed a similar adhesion tendency as ligament cells. These two kinds of cells adhered significantly more on fibronectin-coated surface than on type I collagen-, type II collagen-, vitronectin- and poly- l-lysine-coated surfaces, while the effect was decreased for chondrocytes. Fibronectin showed to be most potential to promote cell adhesion, and thus would be effective to be incorporated into porous scaffolds for tissue engineering of cartilage and ligament.

Analysis of the promoter region of human cartilage oligomeric matrix protein (COMP).

Cartilage oligomeric matrix protein (COMP) is an extracellular matrix protein expressed in cartilage, ligament, and tendon. The importance of COMP in the matrix of these cells is underscored by the discovery that mutations in COMP cause the skeletal dysplasias, pseudoachondroplasia (PSACH) and multiple epiphyseal dysplasia (EDM1). Here, we present the first report on the analysis of the human COMP promoter region in cartilage, ligament, and tendon cells. A 1.7-kb region of the COMP promoter has been cloned and sequenced and no TATA or CAAT boxes were found. Primer extension identified multiple transcription start sites. All four transcription start sites were utilized in chondrocytes with only three of them utilized in tendon and ligament cells. Differential regulation was observed for different parts of this 1.7-kb region with the 370-bp proximal region conveying the strongest promoter activity. The highest activity was observed in tendon and ligament. Finally, we provide evidence that the DNA binding protein SP1 plays a role in the regulation of COMP expression. These results indicate that COMP expression within these cells is regulated in a unique manner that differs from the expression of other extracellular matrix genes.

Activation of nuclear factor κB at the onset of ossification of the spinal ligaments

We examined the correlation between the activation of nuclear factor κB (NFκB), stimulated by environmental factors involving cytokines and growth factors in ligament cells, and the onset of ossification of the spinal ligaments (OSL) or diffuse idiopathic skeletal hyperostosis (DISH). Aseptic samples were taken carefully from non-ossified sites during surgery (75 patients). We carried out preliminary hematoxylin and eosin and toluidine blue staining, using five portions of each specimen, and excluded samples containing chondrocytic, osteoblastic, or inflammatory cells (n = 25). We used specimens from the remaining 50 patients (35 men and 15 women, ranging in age from 45–81 years); average age, 59.5 years (18 nuchal ligament specimens, and 32 yellow ligament specimens). OSL or DISH had occurred in 25 patients, 20 patients were in the non-OSL group (8 with cervical spondylotic myelopathy, and 12 with lumbar canal stenosis), and the remaining 5 samples were collected from patients with injury. For culture study, we used portions of the 14 largest samples from the above 50 patients. We extracted nuclear proteins and cytoplasmic proteins from non-ossified spinal ligaments in 50 patients and detected p65RelA/NFκB by Western blotting. Tumor necrosis factor-α (TNF α), interleukin 1β (IL-1β), platelet-derived growth factor BB (PDGF-BB) and transforming growth factor-β1 (TGF-β1) in cytoplasm were quantified by enzyme-linked immunosorbent assays (ELISA). Cultured cells from the 14 samples were then stimulated with 10, 100, 250, or 500ng/ml of recombinant human (rh)PDGF-B or TGFβ1. A control experiment was performed without rhPDGF-BB or TGFβ1 stimulation. Alkaline phosphatase (ALP) activity was standardized by the DNA content of the cells. The number of NFκB-positive samples was significantly higher in patients with OSL or DISH than in non-OSL patients. This tendency was obvious in the case of OSL or DISH with non-insulin-dependent diabetes mellitus (NIDDM). In OSL and in DISH patients, significantly greater amounts of PDGF-BB and TGFβ1 were seen in ligament cells than in non-OSL patients (P < 0.05). There was a positive correlation between the detection of p65RelA/NFκB band and the content of PDGF-BB and TGFβ1 in ligament cells (P < 0.05). ALP activity tended to be higher in cells in the OSL group not receiving any other treatment. Our results indicate the possibility that NFκB, stimulated by environmental factors involving PDGF-BB and TGFβ1 in ligament cells, influences the osteoblastic differentiation of undifferentiated mesenchymal cells.

Stimulation of prostaglandin E2 and interleukin-1beta production from old rat periodontal ligament cells subjected to mechanical stress.

Although the severity of periodontal disease is known to be affected by the age of the host, the pathological role of aging in periodontal disease, especially that attributable to trauma from occlusion, has not been well characterized. Prostaglandin (PG)E2 and interleukin (IL)-1beta are key mediators involved in periodontal diseases, potent stimulators of bone resorption, and are produced by human periodontal ligament (PDL) cells in response to mechanical stress. To investigate age-related changes in the biosynthetic capacity of PGE2 and IL-1beta in PDL cells, we examined the effects of in vivo aging with mechanical tension on PGE2 and IL-1beta expression by rat PDL cells. PDL cells obtained from the incisors of 6-week (young) and 60-week (old) rats were cultured on flexible-bottomed culture plates. The cells were deformed by causing a 9% or 18% increase in surface area at 6 cycles per minute for 1 to 5 days. We found an approximately twofold increase in PGE2 and IL-1beta production by old PDL cells subjected to mechanical tension compared with that by young cells, although the constitutive levels were similar in both. The expression of cyclooxygenase (COX)-2 and IL-1beta mRNA (messenger ribonucleic acid) was enhanced by mechanical tension as determined by use of reverse transcription-polymerase chain reaction (RT-PCR), whereas COX-1 and IL-1beta-converting enzyme mRNA remained unchanged. It is possible that the large amount of PGE2 and IL-1beta produced by PDL cells from an aged host in response to mechanical force may be positively related to the acceleration of alveolar bone resorption.

Novel method for the quantitative assessment of cell migration: a study on the motility of rabbit anterior cruciate (ACL) and medial collateral ligament (MCL) cells.

A novel method of quantitating cell migration has been proposed for the potential utilization of tissue engineered scaffolds. Applying Alt's conservation law to describe the motion of first passage ACL and MCL cells, we have developed a quantitative method to assess innate differences in the motility of cells from these two ligamentous tissues. In this study, first passage ACL and MCL cells were cultured from four mature New Zealand white rabbits. One side of the cell monolayer was scraped completely away to create a wound model. The cell moved into the cell-free area, and cell density profiles were analyzed at 6 h and 12 h. Values of the random motility coefficient (mu) were then estimated by curve fitting the 6 h and 12 h data to a mathematical model, derived from the conservation law of cell flux. During 6 h of incubation in medium supplemented with 1% FBS, MCL cells (mu(MCL) = 4.63 +/- 0.65 X 10(-6) mm(2)/sec) were significantly (p < 0.05) more mobile than ACL cells (mu(ACL) = 2.51 +/- 0.31 X 10(-6) mm(2)/sec). At 12 h, the MCL cells also appeared to move faster (mu(ACL) = 4.39 +/- 0.63 X 10(-6) mm(2)/sec, mu(MCL) = 6.59 +/- 1.47 X 10(-6) mm(2)/sec), but the difference was not statistically significant (p = 0.18). Exposure of the cells to growth factors PDGF-BB or bFGF for 6 h had no significant effect on the migration of the ACL and MCL cells. However, exposure of the ACL cells (p < 0.05) and the MCL cells (p = 0.19) to 1 ng/mL of PDGFBB for 12 h enhanced their migration. Incubation with a high concentration (100 ng/mL) of PDGF-BB or bFGF at concentrations tested (1 or 100 ng/mL) for 12 h, produced little or no migratory stimulation on these ligament cells. Our findings support the previous qualitative observations made by numerous investigators. The novel methodology developed in this study may provide a basis for tissue engineering, and the results may be applied to tissue reconstruction techniques of the knee ligaments.

Characterization of Chemotactic Migration and Growth Kinetics of Canine Knee Ligament Fibroblasts

Migration and proliferation of ligament fibroblasts are essential for the healing of ligament injuries. This study was designed to evaluate the migration of intraarticular (anterior and posterior cruciate) and extraarticular (medial and lateral collateral) ligament fibroblasts in response to cytokines and to determine the effect of cell passage on cell proliferation. Recombinant human platelet-derived growth factor, hepatocyte growth factor/scatter factor, and bone morphogenic protein-2 stimulated the migration of all ligament cells in a dose-dependent manner, with optimal migration at 10 ng/ml. Recombinant human epithelial growth factor preferentially stimulated the migration of intraarticular ligament fibroblasts, whereas recombinant human interleukin-1 was more effective with extraarticular ligament fibroblasts. Recombinant human insulin-like growth factor-1, insulin-like growth factor-2, transforming growth factor-beta, and fibroblast growth factor had no significant effect on the migration of ligament-derived fibroblasts. These data suggest that specific cytokines stimulate the migration of knee ligament fibroblasts and provide a rationale for possible therapeutic approaches to optimize ligament healing. Fibroblasts derived from the anterior cruciate ligament have been shown to proliferate at a slower rate than those derived from the medial collateral ligament. We have extended these observations and have demonstrated that fibroblasts from both the posterior and anterior cruciate ligaments proliferate at a slower rate than lateral and medial collateral ligament-derived fibroblasts. The differences between the growth rates of intraarticular and extraarticular fibroblasts become insignificant with serial passaging of the cells.