Jm Schakenraad

Influence of substratum wettability on the strength of adhesion of human fibroblasts

To determine the strength of adhesion and the detachment mechanisms of fibroblasts from substrata with different wettability, the behaviour of adhered cells was studied in a parallel-plate flow chamber during exposure to shear. Adhered cells were observed in situ, i.e. in the flow chamber, by phase-contrast microscope and images were analysed semiautomatically. Detachment was found to be dependent both on shear stress and time, although a critical shear stress can be found below which no detachment occurs. On all substrata, cells round up before detachment and are approximately spherical immediately before detachment. The strength of adhesion calculated ranged from 0.6-3.5 x 10(-10) N per cell on FEP-Teflon (the least wettable material included) to 9.4 x 10(-9) N per cell for glass (the most wettable). Ease of detachment seemed to decrease with increasing wettability. However, cells reacted more strongly with tissue culture polystyrene (TCPS) than expected on the basis of its wettability, probably due to surface chemistry.

Preparation and characterization of chemical gradient surfaces and their application for the study of cellular interaction phenomena

Chemical gradient surfaces are surfaces with a gradually changing chemistry along their length which is responsible for a position bound variation in physical properties, most notably, the wettability. In this review, methods to prepare (palladium deposition, diffusion technique, density gradient method, gas diffusion technique, radio frequency plasma and corona discharge, poly(vinylene carbonate) hydrolysis) and characterize gradient surfaces are summarized. The number of techniques available to characterize gradient surfaces is effectively limited to the Wilhelmy plate method for wettability characterization, because the spatial resolution of more chemically oriented techniques, like infrared spectroscopy or X-ray photoelectron spectroscopy is still too limited, apart from their poor surface sensitivity as compared to contact angles. Gradient surfaces are especially useful to study biological interactions along their lengths, as the influence of the entire wettability spectrum upon protein adsorption or cellular interactions can be obtained in one single experiment, therewith minimizing biological variations. In general, proteins adsorb more extensively on the hydrophobic ends of gradient surfaces, which is accompanied by a lesser spreading and adhesion of tissue cells than on the hydrophilic ends of gradient surfaces. An influence of the specific chemistry constituting the gradient, upon protein adsorption as well as on cellular interactions always remains, indicating that biological interactions at an interface are not solely governed by wettability.

Light-microscopic and electron-microscopic evaluation of short-term nerve regeneration using a biodegradable poly(DL-lactide-epsilon-caprolacton) nerve guide

The aim of this study was to evaluate short-term peripheral nerve regeneration across a 10-mm. gap, using a biodegradable poly(DL-lactide-epsilon-caprolacton) nerve guide, with an internal diameter of 1.5 mm and a wall thickness of 0.30 mm. To do so, we evaluated regenerating nerves using light microscopy, transmission electron microscopy and morphometric analysis after implantation of 12-mm nerve guides in the sciatic nerve of the rat. Evaluation times ranged from 3-10 weeks. Three weeks after reconstruction, myelinated nerve fibers could be observed in the distal nerve stump. Ten weeks after reconstruction, the regenerating nerves already resembled normal nerves. In conclusion, we show that poly(DL-lactide-epsilon-caprolacton) nerve guides can be successfully applied in the reconstruction of severed nerves in the rat model. Furthermore, we have observed the fastest nerve regeneration described thus far, after reconstruction using a biodegradable nerve guide.

Biodegradable hollow fibres for the controlled release of drugs

Biodegradable hollow fibres of poly-L-lactic acid (PLLA) filled with a suspension of the contraceptive hormone levonorgestrel in castor oil were implanted subcutaneously in rats to study the rate of drug release, rate of biodegradation and tissue reaction caused by the implant. The in vivo drug release was compared with the release in vitro using different release media. Fibres, disinfected with alcohol showed a zero-order release, both in vitro and in vivo, for over 6 months. Fibres, either gamma-sterilized or disinfected with alcohol were harvested at time intervals ranging from 1 d to 6 months after implantation. Molecular weights of PLLA, tensile strengths, and remaining amounts of drug were determined as a function of time. The tissue reaction can be described as a very moderate foreign body reaction with the initial presence of macrophages, which are gradually replaced by fibroblasts which form a collagen capsule. Molecular weight determinations of PLLA showed a decrease from an initial Mw of 1.59 X 10(5) to 5.5 X 10(4) in 4 months (after alcohol sterilization). A gradual decrease in fibre strength with time was observed which did not significantly impair the release rate of levonorgestrel.

A new PLLA/PCL copolymer for nerve regeneration

The aim of this study is to evaluate the functional and cell biological applicability of a two-ply nerve guide constructed of a PLLA/PCL (i.e. poly-l-lactide and poly-ε-caprolactone) copolymer. To do so, we performed a cytotoxicity test, a subcutaneous biodegradation test and an in situ implantation study in the sciatic nerve of the rat. The nerve guide copolymer was found to be non-toxic, according to ISO/EN standards, and it showed a mild foreign body reaction and complete fibrous encapsulation after implantation. Onset of biodegradation of the inner layer was seen after one month of implantation. After 18 months of implantation complete fragmentation was observed, as well as a secondary inflammatory response characterized by foreign body giant cell activity and phagocytosis of polymer debris. Recovery of both motor and sensory nerve function was observed in all nerve guides.

THERMODYNAMIC ASPECTS OF CELL SPREADING ON SOLID SUBSTRATA

To verify the validity of thermodynamic approaches to the prediction of cellular behavior, cell spreading of three different cell types on solid substrata was determined in vitro. Solid substrata as well as cell types were selected on the basis of their surface free energies, calculated from contact angle measurements. The surface free energies of the solid substrata ranged from 18–116 erg cm−2. To measure contact angles on cells, a technique was developed in which a multilayer of cells was deposited on a filter and air dried. Cell surface free energies ranged from 60 erg cm−2 for fibroblasts, and 57 for smooth muscle cells, to 91 for HeLa epithelial cells. After adsorption of serum proteins, cell surface free energies of all three cell types converged to approx 74 erg cm−2. The spreading of these cell types from RPMI 1640 medium on the various solid substrata showed that both in the presence and in the absence of serum proteins in the medium, cells spread poorly on low energy substrata (Ys<50 erg cm−2), whereas good cell spreading was observed on the higher energy substrata. Calculations of the interfacial free energy of adhesion (ΔFadh) show that ΔFadh decreases with increasingYs, and equals zero around 45 erg cm−2 for all three cell types in the presence of serum proteins and for HeLa epithelium cells in the absence of serum proteins. This explains the spreading of these cells on the various substrata upon a thermodynamic basis. The results clearly show that substratum surface free energy has a predictive value with respect to cell spreading in vitro, both in the presence and absence of serum proteins. It is noted, however, that interfacial thermodynamics fail to explain the behavior of fibroblasts and smooth muscle cells in the absence of serum proteins, most likely because of the relatively high surface charges of these two cell types.

Long-term evaluation of degradation and foreign-body reaction of subcutaneously implanted poly(DL-lactide-epsilon-caprolactone)

The aim of this study was to evaluate the degradation and foreign-body reaction of poly(DL-lactide-epsilon-caprolactone) (PLA85CL50) bars. This specific biomaterial is used for the construction of nerve guides, which can be used in the reconstruction of short nerve gaps. Subcutaneously implanted PLA85CL50 bars were harvested after implantation periods ranging from 3 to 12 months and evaluated for the rate of degradation and the degree of foreign-body reaction. It was observed that this copolymer degraded completely within 12 months and that no lactide or epsilon-caprolactone crystals were formed. Furthermore, we conclude that the foreign-body reaction of PLA85CL50 is very mild. These properties make the amorphous copolymer of DL-lactide and epsilon-caprolactone (50:50) suitable for the construction of nerve guides.

Poly(DL-lactide-epsilon-caprolactone) nerve guides perform better than autologous nerve grafts.

The aim of this study was to compare the speed and quality of nerve regeneration after reconstruction using a biodegradable nerve guide or an autologous nerve graft. We evaluated nerve regeneration using light microscopy, transmission electron microscopy and morphometric analysis. Nerve regeneration across a short nerve gap, after reconstruction using a biodegradable nerve guide, is faster and qualitatively better, when compared with nerve reconstruction using an autologous nerve graft. Therefore, we conclude that in the case of a short nerve gap (1 cm), reconstruction should be carried out using a biodegradable nerve guide constructed of a copolymer of DL‐lactide and ϵ‐caprolactone.

Biodegradation of porous versus non-porous poly(L-lactic acid) films

The influence of porosity on the degradation rate of poly(L-lactic acid) (PLLA) films was investigated in vitro and in vivo. Non-porous, porous and “combi” (porous with a non-porous layer) PLLA films were used. Changes in Mw, Mn, polydispersity (Mw/Mn) ratio, melting temperature (Tm), heat of fusion, tensile strength, E-modulus, mass and the remaining surface area of cross-sections of the PLLA films were measured. In general, during the degradation process, the porous film has the highest Mw, Mn, Mw/Mn ratio and Tm, while the non-porous film has the lowest. In contrast, the highest heat of fusion values were observed for the non-porous film, indicating the presence of relatively smaller molecules forming crystalline domains more easily. The tensile strength and E-modulus of the non-porous film decrease faster than those of the porous and the combi film. None of the three types of films showed massive mass loss in vitro nor a significant decrease in remaining polymer surface area in light microscopical sections in vitro and in vivo. Heavy surface erosion of the non-porous layer of the combi film was observed after 180 days, turning the combi film into a porous film. This is also indicated by the changes in tensile strength, Mw, Mw/Mn, Tm and heat of fusion as a function of time. It is concluded that non-porous PLLA degrades faster than porous PLLA. Thus, in our model, porosity is an important determinant of the degradation rate of PLLA films.

In vivo and in vitro degradation of poly[50/50 (85/15L/D)LA/ε-CL], and the implications for the use in nerve reconstruction

Nerve guides can be used for the reconstruction of peripheral nerve defects. After serving their function, nerve guides should degrade. p[50/50 (85/15L/D)LA/e-CL] degrades completely within 1 year without the formation of a slow degrading crystalline fraction. Although the tensile strength (TS) of a p[50/50 (85/15L/D)LA/e-CL] nerve guide is negligible after 2 months, nerve regeneration across a 1-cm gap in the sciatic nerve of the rat is faster and qualitatively better than after reconstruction using autologous nerve grafts. During degradation p[50/50 (85/15L/D)LA/e-CL] swells, especially during the first 3 months. This can have a negative influence on the regenerating nerve. p[50/50 (85/15L/D)LA/e-CL] nerve guides could only be used in the clinical situation in case of short nerve gaps (several mm) in small nerves (for instance digital nerves). Refinements will be needed to successfully reconstruct longer nerve gaps (several cm).