Proliferative and matrix synthesis response of canine anterior cruciate ligament fibroblasts submitted to combined growth factors.

We investigated the effects of growth factors on the proliferation and matrix synthesis of anterior cruciate ligament fibroblasts. Fibroblasts from the anterior cruciate ligaments of dogs were transferred at the second passage in a defined medium. Epidermal growth factor, platelet-derived growth factor-AB, transforming growth factor-beta 1, insulin-like growth factor-1, and insulin, combined two by two following a 5 x 5 logarithmic concentration matrix, were added. Tridimensional curves showing cell proliferation at 24 hours against the concentration of two effectors were obtained for each combination. Collagen and proteoglycan productions were quantified using [14C]glycine and Na2[35S]O4. Ratios of type I:III collagen and hydrodynamic size distributions of proteoglycans were assayed, respectively, by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and gel filtration chromatography. Epidermal growth factor had an effect nearly equivalent to that of platelet-derived growth factor-AB on cell proliferation. Both had a greater effect than insulin-like effect of transforming growth factor-beta 1. Neither platelet-derived growth factor-AB nor insulin has a significant effect by itself on collagen production. Epidermal growth factor slightly decreases collagen production as well as the type I:III collagen ratio; both transforming growth factor-beta 1 and insulin-like growth factor-1 increase the same parameters. Epidermal growth factor inhibits the stimulation induced by transforming growth factor-beta 1. Similarly, insulin decreases the response to insulin-like growth factor-1. Proteoglycan production was significantly increased by all growth factors in this study, with transforming growth factor-beta 1 having the strongest effect. Small hydrodynamic size of proteoglycan was correlated to a high level of proteoglycan. biosynthesis. The results may be readily applied to tissue engineering or provide a basis for in vivo investigations.

Fibroblast seeding and culture in biodegradable porous substrates.

A natural poly(hydroxybutyrate-co-9% hydroxyvalerate) copolyester was processed into a three-dimensional porous foam structure by salt leaching/solvent casting with previously sieved sodium chloride salts. Laboratory-built P(HB-9% HV) foams and commercial collagen sponges were cut into small rectangular specimens, sterilized, and prewetted using ethanol, rinsed with Dulbecco's minimum essential medium + 10% serum culture media, and seeded with fibroblasts isolated from canine anterior cruciate ligaments. The fibroblast cultures into such porous substrates were performed from 0 to 35 days by incubation (5% CO2) at 37 degrees C. It demonstrated that the P(HB-HV) sustained a cell proliferation rate similar to that observed in collagen sponges, up to at least 35 days, with a maximal cell density on the day 28 in culture. On the other hand, the P(HB-HV) materials kept their structural integrity during the culture period while the collagen foams contracted greatly. Further, the total protein production after 4 weeks in culture was found to be twice as high (190 +/- 10%) in the P(HB-9% HV) foam than in the collagen foam. Porous P(HB-HV) materials appear to be adequate polymeric substrates for cell cultures. However, further evaluations are still required to confirm such preliminary results.

[Responses of ligamentous fibroblasts to mechanical stimulation]. / Réponses des fibroblastes ligamentaires à la stimulation mécanique.

Mechanical stimulation, as provided by physiotherapy or controlled motion is essentially the only factor able to improve anterior cruciate ligament (ACL) healing. We investigate the cellular effects of such stimulus. Two types of stimulations are applied on canine ACL fibroblasts: repetitive stretch of an elastomeric adhesion substrate and a laminar flow of culture media over the culture surface. Cell orientation, proliferation rate, synthesis and type of collagen as well as proteoglycans (PG) synthesis and hydrodynamic characteristics have been studied. According to our results, the fibroblasts tend to align perpendicularly to the deformation axis of their substrate, and along a laminar flow. The shear stress induced by the laminar flow does not modify significantly proliferation rate nor extracellular matrix synthesis. Substrate stretching however, increases proliferation rate, collagen synthesis, mostly type III, and PG synthesis, principally of small sizes. The characteristics of fibroblasts submitted to repeated deformation match those of fibroblasts from ligament scar tissues. Their orientation perpendicular to substratum deformation differs from the one usually encountered in the undamaged tissue: aligned on the ligament axis.

[Assays of cytotoxicity of the Nickel-Titanium shape memory alloy]. / Essais de cytotoxicité sur l'alliage à mémoire de forme Nickel-Titane.

The equiatomic Nickel-Titanium (NiTi) alloy has exceptional mechanical properties such as shape memory and superelasticity. It already has applications in orthodontics and is a promising orthopaedic biomaterial. Cytocompatibility studies must therefore be undertaken. The objective of this study is to determine the biological response that NiTi elicits compared to other orthopaedic metals currently used in orthopaedic surgery. Cytotoxicity tests constitute an efficient first step in a biocompatibility study and contribute to reduce animal use in laboratory. Direct contact and agar diffusion cytotoxicity assays were performed following ASTM standards #F813-83 and #F895-84 respectively. Confluent L-929 fibroblasts culture plates were incubated (directly or under an agar bed) in presence of NiTi, titanium (Ti), vitallium (Co-Cr-Mo) and 316L stainless steel discs. Following exposition to specimens, a vital dye was added to the plates. All cultures were evaluated for cytotoxic reactions, under light microscopy. Direct contact and agar diffusion assays indicated that all metals tested induced a mild biological reaction. Specimens were ranked according to an index of biological response, they are enumerated here in decreasing order of cytotoxicity: NiTi approximately Co-Cr-Mo >> pure grade 4 Ti approximately pure grade 1 Ti approximately Ti 6A1 4V approximately 316L stainless steel. Furthermore, plasma surface modification increased the cytocompatibility of NiTi.

Viscoelastic properties of the human lumbodorsal fascia.

The purpose of this study is to provide better understanding of the mechanical response of the lumbodorsal fascia to dynamic and static traction loadings. Since the fascia shows a viscoelastic behaviour, tests in which time is a variable were used, namely hysteresis and stress relaxation. Load-strain and load-time curves obtained from the hysteresis and stress-relaxation tests point out three different phenomena. First, an increase in stiffness is noticed when ligaments are successively stretched, i.e. strains produced by successive and identical loads decrease. Second, if a sufficient resting period is allowed between loadings, stiffening is reversed and strains tend to recover initial values. The third phenomenon, observed in stress-relaxation tests as time progresses, is ligament contraction in stretched and isometrically held samples. This third phenomenon may be explained by the possibility that muscle fibres capable of contracting spontaneously could be present in lumbodorsal fascia ligaments.

[Culture of fibroblasts of canine anterior cruciate ligaments in a defined medium, effect of epidermal growth factors and platelet derived growth factors]. / Culture de fibroblastes de ligaments croisés antérieurs canins en milieu défini, effet des Epidermal Growth Factors et des Platelet Derived Growth Factors.

Wounded ACL heal very poorly. Following ligament rupture, the initial scar tissue is highly unorganized and is mechanically and biochemically very different from the normal tissue. As fibroblasts play the main part in ligament healing and remodelling process, we try to construct a model of fibroblast's response to various environmental conditions. This type of model would provide a solid ground for improving therapies. A fibroblast strain has been isolated from canine ACL. A totally defined, serum-free medium has been optimized for that strain. We adapted and modified the common techniques, using radio-isotopes, for quantifying DNA, collagen and proteoglycan synthesis. Dose-response curves obtained by these methods are given for Epidermal Growth Factors and Platelet Derived Growth Factors. Both factors are mitogenic, PDGF more so than EGF. Collagen production is affected by neither, while PG synthesis is down-regulated by PDGF.

A computer-controlled apparatus for in vitro mechanical stimulation and characterization of ligaments.

A system has been developed to study in vitro the effects of mechanical stimulation on the biomechanical properties of ligaments. The apparatus is based on a ball screw driven by a microcomputer-controlled stepper motor capable of generating 100 Newtons of traction, the resulting force in the tissue is monitored in real-time acquisition by a load cell. It is programmed to perform virtually any kind of mechanical stimulation or biomechanical characterization tests. Preliminary tests on canine anterior cruciate ligaments indicate that this system is adequate for a variety of mechanical stimulations and characterization assays.