In vivo screening of extracellular matrix components produced under multiple experimental conditions implanted in one animal.

Animal experiments help to progress and ensure safety of an increasing number of novel therapies, drug development and chemicals. Unfortunately, these also lead to major ethical concerns, costs and limited experimental capacity. We foresee a coercion of all these issues by implantation of well systems directly into vertebrate animals. Here, we used rapid prototyping to create wells with biomaterials to create a three-dimensional (3D) well-system that can be used in vitro and in vivo. First, the well sizes and numbers were adjusted for 3D cell culture and in vitro screening of molecules. Then, the functionality of the wells was evaluated in vivo under 36 conditions for tissue regeneration involving human mesenchymal stem cells (hMSCs) and bovine primary chondrocytes (bPCs) screened in one animal. Each biocompatible well was controlled to contain µl-size volumes of tissue, which led to tissue penetration from the host and tissue formation under implanted conditions. We quantified both physically and biologically the amounts of extracellular matrix (ECM) components found in each well. Using this new concept the co-culture of hMSCs and bPCs was identified as a positive hit for cartilage tissue repair, which was a comparable result using conventional methods. The in vivo screening of candidate conditions opens an entirely new range of experimental possibilities, which significantly abates experimental animal use and increases the pace of discovery of medical treatments.

Polymer hollow fiber three-dimensional matrices with controllable cavity and shell thickness.

Hollow fibers find useful applications in different disciplines like fluid transport and purification, optical guidance, and composite reinforcement. In tissue engineering, they can be used to direct tissue in-growth or to serve as drug delivery depots. The fabrication techniques currently available, however, do not allow to simultaneously organize them into three-dimensional (3D) matrices, thus adding further functionality to approach more complicated or hierarchical structures. We report here the development of a novel technology to fabricate hollow fibers with controllable hollow cavity diameter and shell thickness. By exploiting viscous encapsulation, a rheological phenomenon often undesired in molten polymeric blends flowing through narrow ducts, fibers with a shell-core configuration can be extruded. Hollow fibers are then obtained by selective dissolution of the inner core polymer. The hollow cavity diameter and the shell thickness can be controlled by varying the polymers in the blend, the blend composition, and the extrusion nozzle diameter. Simultaneous with extrusion, the extrudates are organized into 3D matrices with different architectures and custom-made shapes by 3D fiber deposition, a rapid prototyping tool which has recently been applied for the production of scaffolds for tissue engineering purposes. Applications in tissue engineering and controlled drug delivery of these constructs are presented and discussed.

Stimulation of skin repair is dependent on fibroblast source and presence of extracellular matrix.

In this study in vitro and in vivo functions were compared between cultured dermal equivalents produced with human fibroblasts isolated either from papillary dermis or adipose tissue of the same donors. Papillary dermal fibroblasts had a normal spindle cell shape; in contrast, adipose tissue fibroblasts had a stellate cell shape, actin stress fibers containing alpha-smooth muscle actin, multiple narrow extensions at their edges, and longer focal adhesion plaques. After dynamic culture for 14 days in PEGT/PBT carrier scaffolds, cell numbers between the two cell sources were comparable, but tissue morphology was different between the cultured groups. In addition, papillary fibroblasts had deposited significantly more glycosaminoglycans (214 +/- 15 versus 159 +/- 21 microg, p < 0.001) and a lower amount of collagen (49 +/- 14 versus 111 +/- 25 microg of hydroxyproline, p < 0.001) than had adipose fibroblasts. Moreover, the latter constructs were significantly more contracted than the papillary fibroblast-cultured constructs (78 +/- 6 versus 96 +/- 3%, p < 0.001). In comparison with the influence of cultured dermal equivalents on wound healing, the transplantation of five groups (control acellular carrier, papillary fibroblast-seeded construct, adipose fibroblast-seeded construct, papillary fibroblast-cultured construct, and adipose fibroblast-cultured construct) to full-thickness wounds on the backs of athymic mice showed clear differences in angiogenesis and tissue ingrowth after 10 days, and in reepithelialization after 21 days. After 10 days, the level of vascular ingrowth in the carrier (von Willebrand staining) for the five groups was as follows: adipose fibroblast-cultured > papillary fibroblast-cultured = adipose fibroblast-seeded > papillary fibroblast-seeded > acellular carrier. After 21 days, only the acellular carriers were not vascularized and the papillary fibroblast-seeded constructs were not completely vascularized. Complete wound reepithelialization (92 +/- 12%) was observed only in the group treated with adipose cultured constructs. Wound contraction was not observed. Staining for HLA-ABC and alpha-smooth muscle actin showed that human fibroblasts had survived and that adipose fibroblasts continued to express the actin isoform. These results showed not only stimulation of skin repair when fibroblasts were present in the carrier, but also significant positive effects of the deposited extracellular matrix (ECM) in the carrier. In addition, the adipose fibroblast-seeded construct, and especially the adipose fibroblast-cultured construct, significantly stimulated angiogenesis and reepithelialization when compared with their corresponding papillary fibroblast constructs. Apparently, tissue source or fibroblast phenotype and the presence of ECM play a crucial role in the stimulation of (impaired) healing and engineering of dermal equivalents.