Enzymatic post-crosslinking of printed hydrogels of methacrylated gelatin and tyramine-conjugated 8-arm poly(ethylene glycol) to prepare interpenetrating 3D network structures.

Methacrylated gelatin (GelMA) has been intensively studied as a 3D printable scaffold material in tissue regeneration fields, which can be attributed to its well-known biological functions. However, the long-term stability of photo-crosslinked GelMA scaffolds is hampered by a combination of its fast degradation in the presence of collagenase and the loss of physical crosslinks at higher temperatures. To increase the longer-term shape stability of printed scaffolds, a mixture of GelMA and tyramine-conjugated 8-arm PEG (8PEGTA) was used to create filaments composed of an interpenetrating network (IPN). Photo-crosslinking during filament deposition of the GelMA and subsequent enzymatic crosslinking of the 8PEGTA were applied to the printed 3D scaffolds. Although both crosslinking mechanisms are radical based, they operate without interference of each other. Rheological data of bulk hydrogels showed that the IPN was an elastic hydrogel, having a storage modulus of 6 kPa, independent of temperature in the range of 10 - 40°C. Tensile and compression moduli were 110 kPa and 80 kPa, respectively. On enzymatic degradation in the presence of collagenase, the gelatin content of the IPN fully degraded in 7 days, leaving a stable secondary crosslinked 8PEGTA network. Using a BioMaker bioprinter, hydrogels without and with human osteosarcoma cells (hMG-63) were printed. On culturing for 21 days, hMG-63 in the GelMA/8PEGTA IPN showed a high cell viability (>90%). Thus, the presence of the photoinitiator, incubation with H2O2, and mechanical forces during printing did not hamper cell viability. This study shows that the GelMA/8PEGTA ink is a good candidate to generate cell-laden bioinks for extrusion-based printing of constructs for tissue engineering applications.

Porous hybrid structures based on P(DLLA-co-TMC) and collagen for tissue engineering of small-diameter blood vessels.

Poly (D,L-lactide)-7co-(1,3-trimethylene carbonate) [P(DLLA-co-TMC)] (83 mol % DLLA) was used to produce matrices suitable for tissue engineering of small-diameter blood vessels. The copolymer was processed into tubular structures with a porosity of approximately 98% by melt spinning and fiber winding, thus obviating the need of organic solvents that may compromise subsequent cell culture. Unexpectedly, incubation in culture medium at 37 degrees C resulted in disconnection of the contact points between the polymer fibers. To improve the structural stability of these P(DLLA-co-TMC) scaffolds, a collagen microsponge was formed inside the pores of the synthetic matrix by dip coating and freeze drying. Hybrid structures with a porosity of 97% and an average pore size of 102 mum were obtained. Structural stability was preserved during incubation in culture medium at 37 degrees C. Smooth-muscle cells (SMCs) were seeded in these hybrid scaffolds and cultured under pulsatile flow conditions in a bioreactor (120 beats/min, 80-120 mmHg). After 7 days of culture in a dynamic environment viable SMCs were homogeneously distributed throughout the constructs, which were five times stronger and stiffer than noncultured scaffolds. Values for yield stress (2.8 +/- 0.6 MPa), stiffness (1.6 +/- 0.4 MPa), and yield strain (120% +/- 20%) were comparable to those of the human artery mesenterica.

Biological characterisation of vascular grafts cultured in a bioreactor.

In this study, the development is described of a tissue-engineered construct mimicking the structure of a natural blood vessel. Smooth muscle cells (SMC) were cultured under pulsatile flow conditions in porous tubular scaffolds composed of crosslinked type I insoluble collagen and insoluble elastin. Under these dynamic culture conditions, average wall shear rate, systolic and diastolic pressures and pressure wave-forms comparable to conditions in the human carotid artery were obtained. Culturing of SMC in tubular scaffolds under dynamic conditions resulted in enhanced tissue formation compared to static conditions. Higher SMC numbers, a more homogeneous distribution of SMC throughout the scaffolds and higher collagen mRNA expression levels were found when cells were cultured under dynamic compared to static conditions. mRNA expression levels of markers of proliferation and apoptosis showed that the higher cell numbers in the scaffolds cultured under dynamic conditions can be explained by increased cell proliferation but not by decreased apoptosis. Glucose consumption and lactate formation by the cells showed that cell metabolism was more aerobic under dynamic compared to static conditions. Lining of the dynamically cultured constructs with a luminal monolayer of endothelial cells might result in vessels suitable for in vivo applications.

Biocompatibility and degradation of aliphatic segmented poly(ester amide)s: in vitro and in vivo evaluation.

Aliphatic segmented poly(ester amide)s, comprising a crystallizable amide phase and a flexible amorphous ester phase, were investigated for potential use in biomedical applications. By varying the amide content and the type of crystallizable amide segments, the polymer's thermal and mechanical properties can readily be tuned. Polymers with 25 and 50 mol % of amide content are noncytotoxic and sustain growth of fibroblasts onto polymer films. The in vitro degradation of these polymers was followed in PBS (pH 7.4) at 37 degrees C up to 7 months. The poly(ester amide)s showed the characteristics of bulk degradation with a gradual decrease in molecular weight and almost no mass loss. The in vivo degradation of these polymers, followed by subcutaneous implantation in rats up to 6 weeks, was slow and similar to the in vitro degradation. The tissue response upon implantation was followed over 6 weeks. A mild foreign-body reaction, characterized by the presence of macrophages, and sporadically a lymphocyte, were observed in the first week of implantation. After 6 weeks the implant site is characterized by fibrous encapsulation with no signs of inflammation. The poly(ester amide)s tested are biocompatible, but their in vitro as well as in vivo degradation is very slow.

Physical characterization of vascular grafts cultured in a bioreactor.

Tubular scaffolds of collagen and elastin (weight ratio 1:1) with interconnected pores were prepared by freeze drying and crosslinked with N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) in the presence or absence of a Jeffamine spacer (poly(propylene glycol)-bis-(2-aminopropyl ether), J230). The crosslinked and uncrosslinked matrices had porosities of 90% and average pore sizes of 131-151 microm. Smooth muscle cells (SMC) were cultured in the crosslinked and uncrosslinked tubular scaffolds under pulsatile flow conditions (mean flow rate 9.6 ml/min, 120 beats/min, pressure 80-120 mmHg). All the constructs could withstand cyclic mechanical strain in the absence of any mechanical support without cracking or suffering permanent deformation. After 7d, SMC were homogeneously distributed throughout the uncrosslinked and EDC/NHS crosslinked constructs, whereas hardly any cell was observed on the luminal side of J230/EDC/NHS crosslinked matrices. Considering the better mechanical performance of EDC/NHS crosslinked matrices compared to non-crosslinked constructs after 7d of culture, SMC were dynamically cultured in the former scaffolds for 14d. During this period, the high strain stiffness of the constructs increased more than two-fold to 38+/-2 kPa, whereas the low strain stiffness doubled to 8+/-2 kPa. The yield stress and yield strain were 30+/-10 kPa and 120+/-20%, respectively. SMC were homogeneously distributed throughout the EDC/NHS crosslinked collagen/elastin constructs and collagen fibres tended to orient in the circumferential direction.