R. Olkowski

Development of a pre-vascularized 3D scaffold-hydrogel composite graft using an arterio-venous loop for tissue engineering applications

Hyaluronic acid (HA) and fibrin glue (FG) are effective hydrogels for tissue engineering applications as they support tissue in-growth, retain growth factors, and release them slowly with time. The scaffolds, in combination with a hydrogel, effectuate a successful graft. However, the survival of a graft entirely depends upon a functional vascular supply. Therefore, hydrogels must support the in-growing vasculature. To study and compare the vascular patterns, HA and FG hydrogel-containing PLDLLA-TCP-PCL scaffolds were implanted in the groin of male Lewis rats and supplied with a micro-surgically prepared arterio-venous (A-V) loop. The rats were perfused with a vascular contrast media after 4 and 8 weeks and sacrificed for further analysis. The specimens were scanned with micro-CT to find the vascular growth patterns. Corrosion casting of blood vessels followed by SEM demonstrated a high vascular density near the parent blood vessels. Histologically, HA and FG implanted animal groups showed significant angiogenetic activity, especially within the pores of the scaffold. However, formation of new blood vessels was more conspicuously observed at 4 weeks in FG than HA implants. Furthermore, by 8 weeks, the number and pattern of blood vessels were comparable between them. At this time, HA was still present indicating its slow degradation. The finding was confirmed by histomorphometric analysis. This experimental study demonstrates that HA containing composite scaffold systems permit stabile in-growth of blood vessels due to sustained degradation over 8 weeks. HA is a potential matrix for a tissue engineered composite graft.

Cytocompatibility of the selected calcium phosphate based bone cements: comparative study in human cell culture

Calcium phosphate cements (CPC) are valuable bone fillers. Recently they have been also considered as the basis for drug-, growth factors- or cells-delivery systems. Broad possibilities to manipulate CPC composition provide a unique opportunity to obtain materials with a wide range of physicochemical properties. In this study we show that CPC composition significantly influences cell response. Human bone derived cells were exposed to the several well-characterized different cements based on calcium phosphates, magnesium phosphates and calcium sulfate hemihydrate (CSH). Cell viability assays, live/dead staining and real-time observation of cells in contact with the materials (time-laps) were performed. Although all the investigated materials have successfully passed a standard cytocompatibility assay, cell behavior in a direct contact with the materials varied depending on the material and the experimental system. The most recommended were the α-TCP-based materials which proved suitable as a support for cells in a direct contact. The materials which caused a decrease of calcium ions concentration in culture induced the negative cell response, however this effect might be expected efficiently compensated in vivo. All the materials consisting of CSH had negative impact on the cells. The obtained results strongly support running series of cytocompatibility studies for preclinical evaluation of bone cements.

Porous polymeric scaffolds for bone regeneration

Abstract Solvent casting/particulate leaching has been used to synthesize highly porous polymeric scaffolds of controlled pore size, based on poly(methyl methacrylate) (PMMA) and poly(ε-caprolactone) (PCL). Obtained structures have a total porosity of c. 60%, with good interconnections between the pores. Porous scaffolds prepared using the greatest size of NaCl particles have the best mechanical properties. Both PMMA- and PCL-based materials can be sterilized by ionizing radiation. In the case of PCL-based scaffolds, irradiation causes cross-linking of polymer chains, which leads to an improvement of the mechanical properties of the scaffold. The compressive elastic modulus for non-porous samples increases with irradiation dose from 1.5 MPa for 0 kGy to 1.9 MPa for 280 kGy. Preliminary in vitro studies indicate good biocompatibility of both materials.

How calcite and modified hydroxyapatite influence physicochemical properties and cytocompatibility of alpha-TCP based bone cements

Nowadays successful regeneration of damaged bone tissue is a major problem of the reconstructive medicine and tissue engineering. Recently a great deal of attention has been focused on calcium phosphate cements (CPCs) as the effective bone fillers. Despite a number of studies regarding CPCs, only a few compare the physicochemical and biological properties of α-TCP based materials of various phase compositions. In our study we compared the effect of several components (calcite, hydroxyapatite doped with Mg2+, CO32− or Ag+ ions, alginate, chitosan and methylcellulose) on the physicochemical and biological properties of α-TCP-based bone cements. The influence of materials composition on their setting times, microstructure and biochemical stability in simulated body fluid was determined. A number of in vitro laboratory methods, including ICP-OES, metabolic activity test, time-lapse microscopic observation and SEM observations were performed in order to assess biocompatibility of the studied biomaterials. The positive outcome of XTT tests for ceramic extracts demonstrated that all investigated cement-type composites may be considered cytocompatible according to ISO 10993-5 standard. Results of our research indicate that multiphase cements containing MgCHA, AgHA and calcite combined with αTCP enhanced cell viability in comparison to material based only on αTCP. Furthermore materials containing chitosan and methylcellulose possessed higher cytocompatibility than those with alginate.Graphical abstract

Biocompatibility, osteo-compatibility and mechanical evaluations of novel PLDLLA/TcP scaffolds

For tissue engineering of small-diameter blood vessels, biodegradable, flexible and elastic porous tubular structures are most suited. In this study, we prepared crosslinked porous tubular structures from poly(trimethylene carbonate) (PTMC), in which smooth muscle cells (SMCs) were seeded and cultured in a pulsatile bioreactor mimicking the physiological conditions. PTMC was synthesized and porous tubular structures were prepared by dipping coating, cross-linking by g-irradiation, and leaching. SMCs were seeded into the porous structures by perfusion and then the constructs were cultured in a pulsatile bioreactor system. The morphologies, mechnical properties were analyzed and SMCs attachment and proliferation were evaluated by histology studies and CyQuant. Flexible tubular structures were obtained by dip coating with 3mm inner diameter and 1mm wall thickness. The porosity of the structures in wet state reached 85 vol% and the pore sizes were 60-150 mm. PTMC tubular structures showed comparable tensile strength and higher elongation compared with natural blood vessels. A pulsatile bioreactor system mimicking the conditions in vivo (dynamic pressure 70 mmHg, 75 beats/min) was successfully built. Experiements showed 7-day dilation was <10% and variation of diameter at each pulse was <1%. SMCs were homogeneously seeded in the porous scaffolds by perfusion. SMCs proliferate well to form confluent cell layer during a time period of up to 14 days, leading to constructs with even better mechanical performance. PTMC Porous tubular structures were prepared with good microstructures, elasticity and biocompatibility. SMCs were seeded and proliferated well in pulsatile bioreactor system and significant improvement of mechnical strength was observed.