Evi Lippens

Correlation Between Cryogenic Parameters and Physico-Chemical Properties of Porous Gelatin Cryogels

In the present work, we have performed an in-depth physico-chemical and bio-physical evaluation of a series of previously described porous gelatin scaffolds (S. VanVlierberghe, V. Cnudde, P. Dubruel, B. Masschaele, A. Cosijns, I. DePaepe, P.J.S. Jacobs, L. VanHoorebeke, J.P. Remon and E. Schacht, Biomacromolecules 8, 331 (2007)). All scaffolds were prepared by a cryogenic treatment and subsequent freeze-drying. Three types of scaffolds were prepared by using different gelatin concentrations and cooling protocols. Type-I hydrogels were composed of cone-like pores with decreasing diameter from top (330 μm) to bottom (20–30 μm). Type-II and type-III scaffolds contained spherical pores with an average diameter of 135 (type II) and 65 μm (type III), respectively. The physico-chemical and bio-physical properties studied include the water uptake capacity and kinetics, the mechanical properties and the enzyme-mediated degradation. We can conclude that the pore geometry affects the water uptake capacity, the mechanical properties and the degradation profile of the hydrogels. Type-I hydrogels possess the highest water uptake, the lowest compression modulus and the fastest enzyme mediated degradation, indicating a clear effect of the pore morphology (elongated channels for type I versus spherical pores for types II and III) on the physico-chemical and bio-physical properties of the materials. In contrast to the effect of the pore geometry (channel-like versus spherical), the pore size does not significantly affect the water uptake, the mechanical properties and the enzyme mediated degradation in the investigated pore size range (65–135 μm). To the best of our knowledge, this is the first report in which the effects of a cryogenic treatment on the hydrogel network properties are investigated in such detail.

Evaluation of an Injectable, Photopolymerizable, and Three-Dimensional Scaffold Based on Methacrylate-Endcapped Poly(D,L-Lactide-co-ɛ-Caprolactone) Combined with Autologous Mesenchymal Stem Cells in a Goat Tibial Unicortical Defect Model

An in situ crosslinkable, biodegradable, methacrylate-endcapped poly(D,L-lactide-co-epsilon-caprolactone) in which crosslinkage is achieved by photoinitiators was developed for bone tissue regeneration. Different combinations of the polymer with bone marrow-derived mesenchymal stem cells (BMSCs) and alpha-tricalcium phosphate (alpha-TCP) were tested in a unicortical tibial defect model in eight goats. The polymers were randomly applied in one of three defects (6.0 mm diameter) using a fourth unfilled defect as control. Biocompatibility and bone-healing characteristics were evaluated by serial radiographies, histology, histomorphometry, and immunohistochemistry. The results demonstrated cell survival and proliferation in the polymer-substituted bone defects. The addition of alpha-TCP was associated with less expansion and growth of the BMSCs than other polymer composites.

Design and fabrication of a low cost implantable bladder pressure monitor

In the frame of the Flemish Community funded project Bioflex we developed and fabricated an implant for short term (< 7 days) bladder pressure monitoring, and diagnosis of incontinence. This implant is soft and flexible to prevent damaging the bladder’s inner wall. It contains a standard flexible electronic circuit connected to a battery, which are embedded in surface treated silicone to enhance the biocompatibility and prevent salt deposition. This article describes the fabrication of the pill and the results of preliminary cytotoxicity tests. The electronic design and its tests, implantation and the result of the in-vivo experimentation will be presented in other articles.

Cell survival and proliferation after encapsulation in a chemically modified Pluronic® F127 hydrogel

Pluronic® F127 is a biocompatible, injectable, and thermoresponsive polymer with promising biomedical applications. In this study, a chemically modified form, i.e., Pluronic ALA-L with tailored degradation rate, was tested as an encapsulation vehicle for osteoblastic cells. UV cross-linking of the modified polymer results in a stable hydrogel with a slower degradation rate. Toxicological screening showed no adverse effects of the modified Pluronic ALA-L on the cell viability. Moreover, high viability of embedded cells in the cross-linked Pluronic ALA-L was observed with life/death fluorescent staining during a 7-day-culture period. Cells were also cultured on macroporous, cross-linked gelatin microbeads, called CultiSpher-S® carriers, and encapsulated into the modified cross-linked hydrogel. Also, in this situation, good cell proliferation and migration could be observed in vitro. Preliminary in vivo tests have shown the formation of new bone starting from the injected pre-loaded CultiSpher-S® carriers.

Immunohistochemical analysis of low-temperature methylmethacrylate resin-embedded goat tissues.

Goats are frequently used as a suitable animal model for tissue engineering. Immunohistochemistry can be helpful in improving the understanding and evaluation of the in vivo tissue responses at a molecular level. Several commercially available antibodies (KI67, vimentin, CD31, core‐binding factor alpha‐1, osteocalcin, alkaline phosphatase, MAC387, CD3, CD20, CD20cy, CD79 and CD45) were evaluated on Technovit 9100 New© embedded goat tissues. Only vimentin, osteocalcin, MAC387 and CD3 revealed positive staining. These antibodies can be routinely used to evaluate goat tissues at molecular level. The use and development of alternative antibodies might further supplement and complete the possibilities for immunohistochemical analysis of goat tissue samples.

Slow Cooling Cryopreservation of Cell-Microcarrier Constructs

Ideally, tissue engineered constructs should be readily available to meet the need for fast intervention in complex bone defects. To circumvent the long culture period of these constructs before implantation, we investigated the possibility of cryopreserving cell-loaded constructs. Goat bone marrow-derived mesenchymal stem cells (BMSC) and mouse osteoblast-like cells from the MC3T3-E1 cell line were cultured on gelatin CultiSpher-S® microcarriers. These constructs were cryopreserved using the slow cooling technique, i.e. cooling to –80°C at a rate of –1.5°C/min, and were then stored in liquid nitrogen for 1 week. Four different cryomedia were tested, i.e. 90 vol% serum with 10 vol% dimethylsulphoxide (Me2SO) with or without ascorbic acid (AA) and 90 vol% serum supplemented with 5 vol% Me2SO and 5 vol% hydroxyethyl starch or 5 vol% sucrose (60 mM). Cell viability on the constructs was assessed with fluorescent live/dead staining and the colorimetric MTS assay. Cell viability was compared before freezing and at fixed time points after thawing. Immediately after thawing, the viability percentages in all groups were significantly lower than before cryopreservation (p = 0.0369). No significant differences were observed between the viability percentages on the cell constructs cryopreserved in the different media; however, there was a general tendency for higher cell survival and faster recolonization of constructs cryopreserved in Me2SO with or without AA than of the constructs cryopreserved in the other media. For constructs cryopreserved in 10 vol% Me2SO with or without AA, the recolonization period was 3 days for the BMSC constructs and 3.6 and 3.8 days, respectively, for the MC3T3-E1 constructs.

Biocompatibility properties of surface-modified poly(dimethylsiloxane) for urinary applications.

An electronic sensor system for urinary bladder pressure monitoring requires an imbedding into a biocompatible, flexible, and liquid-impermeable material. Poly(dimethylsiloxane) (PDMS) was selected in the present set-up as packaging material because it fulfills the abovementioned requirements. However, the surface of PDMS is hydrophobic and causes undesired interactions with salts, proteins, and cells present in urine. To reduce possible interactions of urine salts in the urinary bladder, monomers, [2-(methacryloyloxy)ethyl]-dimethyl-3-sulfopropyl-ammonium hydroxide (sulfobetaine) and 2-acrylamido-2-methylpropyl sulfonic acid, were grafted onto the surface through oxygen plasma treatment. A reduction in salt deposition between the pure PDMS and the modified PDMS was observed both in vitro (artificial urine flow over the surface) and in vivo (implants into the urinary bladder of experimental pigs). Additionally, a 10-fold reduction in salt deposition was observed in vitro due to grafting of the monomers onto the surface. These modified PDMS materials proved also to be biocompatible in cell cultures, which was further confirmed by histological screening of the bladder tissue after implantation in an in vivo pig model.

In vitro cytotoxicity testing and the application of elastic interconnection technology for short-term implantable electronics

An electronic device was fabricated consisting of 2 flexible electronic circuit islands, interconnected by a 7 cm long elastic interconnection, which could be elongated for at least 50%. This interconnection was based on gold conductor tracks following a 2-D spring pattern, embedded in a biocompatible silicone elastomer. The complete device was embedded in the same silicone elastomer. An in vitro cytotoxicity extraction test, executed on small test-samples in accordance with the ISO 10 993-1 guidelines, revealed that the applied silicone encapsulation to these samples functioned as a good seal for at least 8 days.