Enzymatic activity toward poly(L-lactic acid) implants

Tissue reactions toward biodegradable poly(L-lactic acid) implants were monitored by studying the activity pattern of seven enzymes as a function of time: alkaline phosphatase, acid phosphatase, -naphthyl acetyl esterase, -glucuronidase, ATP-ase, NADH-reductase, and lactate dehydrogenase. Cell types were identified by their specific enzyme patterns, their morphology and location. Special attention was paid to the enzyme patterns of macrophages, fibroblasts and polymorphonuclear granulocytes (PMNs), being involved in foreign body reactions or inflammatory responses. One day after implantation, an influx of neutrophilic and eosinophilic granulocytes was observed, coinciding with activity of alkaline phosphatase (PMN's) and -glucuronidase (eosinophils). From day 3 on, macrophages containing ATP-ase, acid phosphatase and esterase could be observed. From day 7 on, lactate dehydrogenase, the enzyme normally involved in the conversion of lactic acid, and its coenzyme NADH-reductase were observed in macrophages and fibroblasts. These two enzymes demonstrated more activity than expected on basis of wound-healing reactions upon implantation of a nonbiodegradable, inert biomaterial (as, e.g., Teflon). It is concluded that the biodegradable poly (L-lactic acid) used in these implantation studies is tissue compatible, and evokes a foreign body reaction with minor macrophage and giant cell activity, as observed during this 3-week implantation period. Most enzyme patterns were simply due to a wound-healing reaction. The slightly increased levels of LDH and NADH suggest the release of lactic acid from the implant, and thus confirms the biodegradable nature of this polymer

Changes in the mechanical properties of dermal sheep collagen during in vitro degradation

The changes in tensile strength, elongation at break, and high strain modulus of dermal sheep collagen (DSC) during in vitro degradation using bacterial collagenase were studied. The changes in mechanical properties were compared with the change in weight of the samples as a function of degradation time. DSC was crosslinked with either glutaraldehyde (GA) or hexamethylene diisocyanate (HMDIC). During degradation, the changes in mechanical properties of the N-DSC, H-DSC or G-DSC samples were more pronounced than the changes in the weight of the samples. Of the mechanical properties studied, the tensile strength was most susceptible to degradation of the DSC samples. After 2.5 h, N-DSC samples had lost only 55% of their initial weight, but the samples had no tensile strength left. Similar results were obtained for H-DSC, which retained no tensile strength after 24 h degradation, whereas only 45% of the initial weight was lost. G-DSC lost 3.5% of its weight after 24 h degradation, but only 25% of the initial tensile strength remained

Influence of ethylene oxide gas treatment on the in vitro degradation behavior of dermal sheep collagen

The influence of ethylene oxide gas treatment on the in vitro degradation behavior of noncrosslinked, glutaraldehyde crosslinked or hexamethylene diisocyanate crosslinked dermal sheep collagen (DSC) using bacterial collagenase is described. The results obtained were compared with the degradation behavior of either nonsterilized or γ-sterilized DSC. Upon ethylene oxide sterilization, reaction of ethylene oxide with the free amine groups of DSC occurred, which resulted in a decreased helix stability, as indicated by a lowering of the shrinkage temperature of all three types of DSC. Except for the low strain modulus the mechanical properties of the ethylene oxide sterilized materials were not significantly altered. γ-Sterilization induced chain scission in all three types of DSC, resulting in a decrease of both the tensile strength and the high strain modulus of noncrosslinked and crosslinked DSC. When exposed to a solution of bacterial collagenase, ethylene oxide sterilized materials had a lower rate of degradation compared with nonsterilized DSC. This has been explained by a reduced adsorption of the collagenase onto the collagen matrix as a result of the introduction of pendant N-2-hydroxy ethyl groups

Secondary cytotoxicity of (crosslinked) dermal sheep collagen during repeated exposure to human fibroblasts

We investigated commercially available dermal sheep collagen either cross-linked with hexamethylenediisocyanate, or cross-linked with glutaraldehyde. In previous in vitro studies we could discriminate primary, i.e. extractable, and secondary cytotoxicity, due to cell-biomaterial interactions, i.e. enzymatic actions. To develop dermal sheep collagen for clinical applications, we focused in this study on the release, e.g. elimination, of secondary cytotoxicity over time. We used the universal 7 d methylcellulose cell culture with human skin fibroblasts as a test system. Hexamethylenediisocyanate-cross-linked dermal sheep collagen and glutaraldehyde-cross-linked dermal sheep collagen were tested, with intervals of 6 d, over a culture period of 42 d. With hexamethylenediisocyanate-cross-linked dermal sheep collagen, cytotoxicity, i.e. cell growth inhibition and deviant cell morphology, was eliminated after 18 d of exposure. When testing glutaraldehyde-cross-linked dermal sheep collagen, the bulk of cytotoxic products was released after 6 d, but a continuous low secondary cytotoxicity was measured up to 42 d. As a control, non-cross-linked dermal-sheep collagen was tested over a period of 36 d, but no secondary cytotoxic effects were observed. The differences in release of secondary cytotoxicity between hexamethylenediisocyanate-cross-linked dermal sheep collagen, glutaraldehyde-cross-linked dermal sheep collagen and non-cross-linked dermal sheep collagen are explained from differences in cross-linking agents and cross-links obtained. We hypothesize that secondary cytotoxicity results from enzymatic release of pendant molecules from hexamethylene-diisocyanate-cross-linked dermal sheep collagen, e.g. formed after reaction of hydrolysis products of hexamethylenediisocyanate with dermal sheep collagen. Glutaraldehyde-cross-linked dermal sheep collagen contains residual cross-linking agents, which induce the bulk cytotoxicity. Apart from being sensitive to enzymatic degradation, glutaraldehyde-cross-linked dermal sheep collagen was also found to be sensitive to aqueous hydrolysis. Hydrolysis of cross-links may release cytotoxic products and introduce new pendant molecules within glutaraldehyde-cross-linked dermal sheep collagen, which in turn induce cytotoxicity after enzymatic attack

Crosslinking of dermal sheep collagen using hexamethylene diisocyanate

The use of hexamethylene diisocyanate (HMDIC) as a crosslinking agent for dermal sheep collagen (DSC) was studied. Because HMDIC is only slightly water soluble, a surfactant was used to obtain a clear and micellar crosslinking solution and to promote the penetration of HMDIC in the DSC matrix. Using optimized conditions treatment of non-crosslinked DSC (N-DSC) with HMDIC (H-DSC) increased the shrinkage temperature (Ts) of N-DSC from 56°C to 74°C for H-DSC. A linear relation between the decrease in free amine group content and the increase in Ts was observed. Crosslinking with HMDIC did not influence the tensile strength of the N-DSC samples but increased the elongation at break from 141% to 163% and decreased the high-strain modulus from 26 MPa to 16 MPa for the H-DSC samples, respectively

Cross-linking of dermal sheep collagen using a water-soluble carbodiimide

A cross-linking method for collagen-based biomaterials was developed using the water-soluble carbodiimide 1-ethyl-3-(3-dimethyl aminopropyl)carbodiimide hydrochloride (EDC). Cross-linking using EDC involves the activation of carboxylic acid groups to give O-acylisourea groups, which form cross-links after reaction with free amine groups. Treatment of dermal sheep collagen (DSC) with EDC (E-DSC) resulted in materials with an increased shrinkage temperature (Ts) and a decreased free amine group content, showing that cross-linking occurred. Addition of N-hydroxysuccinimide to the EDC-containing cross-linking solution (E/N-DSC) increased the rate of cross-linking. Cross-linking increased the Ts of non-cross-linked DSC samples from 56 to 73 °C for E-DSC and to 86 °C for E/N-DSC samples, respectively. For both cross-linking methods a linear relation between the decrease in free amine group content and the increase in Ts was observed. The tensile strength and the high strain modulus of E/N-DSC samples decreased upon cross-linking from 18 to 15MPa and from 26 to 16MPa, respectively. The elongation at break of E/N-DSC increased upon cross-linking from 142 to 180%

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 γ-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.\ud \ud 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 1.59x10 5 to 5.5 × 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

Adhesion and spreading of cultured endothelial cells on modified and unmodified poly(ethylene terephthalate): a morphological study

The in vitro adhesion and spreading of human endothelial cells (HEC) on hydrophobic poly(ethylene terephthalate) (PETP) and moderately wettable tissue culture polyethylene terephthalate) (TCPETP) were studied with light microscopy and electron microscopy. Numbers of HEC adhering on TCPETP were always higher than those found on PETP. When cells were seeded in the presence of serum, extensive cell spreading on both PETP and TCPETP was observed after the first 30 min. Thereafter, spread cells appeared to withdraw from the PETP surface, resulting in irregularly shaped cells. Complete cell spreading occurred on TCPETP. Complete cell spreading also occurred on PETP and TCPETP when HEC had first been seeded from phosphate buffer solution and serum was supplied after 30 min. Furthermore, HEC spread on both PETP and TCPETP when the surfaces were precoated with protein(s), which promotes cell adhesion. However, when plasma was used for the coating, spread cells did not proliferate in a monolayer pattern. This study shows that TCPETP is, in general, a better surface for adhesion and proliferation of HEC than is PETP, suggesting that vascular prostheses with a TCPETP-like surface will perform better in vivo than prostheses made of PETP