Katrien M. Brouwer

Evaluation of methods for the construction of collagenous scaffolds with a radial pore structure for tissue engineering

Type I collagen is used widely as a biomaterial. The structure of collagenous biomaterials, including pore sizes and general architecture, can be varied by a number of techniques. In this study, we developed a method to construct flat fibrillar type I collagen scaffolds, 6 cm in diameter and with a radially orientated pore structure, by the use of directional freezing. Different methodologies were tested, the optimal one being freezing of a collagen suspension inside‐out, using a centrally positioned liquid nitrogen‐cooled tube. Pore sizes could be varied by the use of different tube materials. Use of aluminium tubes resulted in radial scaffolds with a pore size of 20–30 µm, whereas use of stainless steel produced radial scaffolds with 70–100 µm pore sizes. Brass‐ and copper‐based tubes produced scaffolds with less homogeneous radial pores, pore sizes being 90–100 and 50–80 µm, respectively. Fibreglass tubes gave even less uniformity (pore size 100–150 µm). Scaffolds were free of cracks, except in case of aluminium. Scaffolds with a radial inner structure may be especially suitable for tissue engineering of organs with a radial scaffold structure, such as the diaphragm. Copyright

Regenerative Medicine for the Respiratory System: Distant Future or Tomorrow's Treatment?

Regenerative medicine (RM) is a new field of biomedical science that focuses on the regeneration of tissues and organs and the restoration of organ function. Although regeneration of organ systems such as bone, cartilage, and heart has attracted intense scientific research over recent decades, RM research regarding the respiratory system, including the trachea, the lung proper, and the diaphragm, has lagged behind. However, the last 5 years have witnessed novel approaches and initial clinical applications of tissue-engineered constructs to restore organ structure and function. In this regard, this article briefly addresses the basics of RM and introduces the key elements necessary for tissue regeneration, including (stem) cells, biomaterials, and extracellular matrices. In addition, the current status of the (clinical) application of RM to the respiratory system is discussed, and bottlenecks and recent approaches are identified. For the trachea, several initial clinical studies have been reported and have used various combinations of cells and scaffolds. Although promising, the methods used in these studies require optimization and standardization. For the lung proper, only (stem) cell-based approaches have been probed clinically, but it is becoming apparent that combinations of cells and scaffolds are required to successfully restore the lungs architecture and function. In the case of the diaphragm, clinical applications have focused on the use of decellularized scaffolds, but novel scaffolds, with or without cells, are clearly needed for true regeneration of diaphragmatic tissue. We conclude that respiratory treatment with RM will not be realized tomorrow, but its future looks promising.

Repair of surgically created diaphragmatic defect in rat with use of a crosslinked porous collagen scaffold.

Large defects in congenital diaphragmatic hernia are closed by patch repair, which is associated with a high complication risk and reherniation rate. New treatment modalities are warranted. We evaluated the feasibility of using an acellular biodegradable collagen bioscaffold for a regenerative medicine approach to close a surgically created diaphragmatic defect in a rat model. Scaffold degradation, cellular ingrowth and regeneration of the diaphragm were studied. In 25 rats, a subcostal incision was made and one third of the right hemidiaphragm was resected. Crosslinked porous type I collagen scaffolds (Ø ~ 14 mm) were sutured into the lesion. Rats were sacrificed at 2, 4, 8, 12 or 24 weeks after scaffold implantation. Implants were evaluated macroscopically and (immuno)histologically. Survival after surgery was 88% with no evidence of reherniation. Histological examination showed that the collagen scaffold degraded slowly and new collagen, elastin and mesothelium were deposited. Blood vessels were observed primarily at the outer borders of the scaffold; their number gradually increased in time. Muscle fibres were found on the scaffold covering up to 10% of the defect. Macroscopically, adhesion of the scaffold to the liver was observed. Use of a collagen scaffold to close a surgically created diaphragmatic defect is feasible, with evidence of new tissue formation. The use of crosslinked collagen scaffolds allows targeted modification; e.g. addition of growth factors to further stimulate growth of muscle cells. Copyright

Heparinized collagen scaffolds with and without growth factors for the repair of diaphragmatic hernia: construction and in vivo evaluation.

A regenerative medicine approach to restore the morphology and function of the diaphragm in congenital diaphragmatic hernia is especially challenging because of the position and flat nature of this organ, allowing cell ingrowth primarily from the perimeter. Use of porous collagen scaffolds for the closure of surgically created diaphragmatic defects in rats has been shown feasible, but better ingrowth of cells, specifically blood vessels and muscle cells, is warranted. To stimulate this process, heparin, a glycosaminoglycan involved in growth factor binding, was covalently bound to porous collagenous scaffolds (14%), with or without vascular endothelial growth factor (VEGF; 0.4 µg/mg scaffold), hepatocyte growth factor (HGF; 0.5 µg/mg scaffold) or a combination of VEGF + HGF (0.2 + 0.5 µg/mg scaffold). All components were located primarily at the outside of scaffolds. Scaffolds were implanted in the diaphragm of rats and evaluated after 2 and 12 weeks. No herniations or eventrations were observed, and in several cases, growth factor-substituted scaffolds showed macroscopically visible blood vessels at the lung site. The addition of heparin led to an accelerated ingrowth of blood vessels at 2 weeks. In all scaffold types, giant cells and immune cells were present primarily at the liver side of the scaffold, and immune cells and individual macrophages at the lung side; these cell types decreased in number from week 2 to week 12. The addition of growth factors did not influence cellular response to the scaffolds, indicating that further optimization with respect to dosage and release profile is needed.