Flow Perfusion Culture With Fibroblast-seeded Plla-collagen Scaffolds For Abdominal Wall Repair

No ideal surgical management technique to reconstruct large abdominal wall defects has as yet been identified

. Complication rates in these complex cases, regardless of closure technique, have been reported to be in excess of 90%.

The use of absorbable meshes as a bridge to definitive hernia repair has been considered, but their use currently requires multiple surgeries which carries the risk of increased morbidity. Use of permanent meshes is contraindicated in contaminated surgical fields, which are common in these cases.

Autologous tissue repair has been suggested as a possible alternative in these cases. However, any repair is limited by the size of the defect and the integrity of the abdominal wall. Biologically-derived, acellular matrix grafts have shown successful abdominal wall reconstruction and resistance to infection in contaminated cases, but they are generally only slowly remodelled as a neotissue or are degraded too quickly.

Matrices seeded with cells for urological repair have demonstrated increased tissue regeneration and mechanical performance compared to acellular scaffolds, and it is possible that a similar technological approach could provide the required advance for this surgical procedure.

Dermal fibroblasts constitute a potential autogenous source of cells for abdominal wall tissue engineering because they play a key role ingrowth factor secretion, matrix deposition, and matrix degradation. Fibroblasts participate in wound healing by their ability to secrete prodigious quantities of ECM proteins and responding to and synthesizing cytokines, chemokines, and other mediators of inflammation.

In addition, populations of fibroblasts have been shown to differentiate to become myofibroblasts that can exert contractile force due to the expression of myosin and alpha-smooth muscle actin, enabling wound area reduction. Fibroblast isolates have also been shown to contain progenitors able to differentiate into neurons and muscle cells in vitro that show high levels of plasticity.

Our hypothesis is that by using highly proliferative cells with a unique scaffold, we would be able to accelerate the regeneration of abdominal wall repair tissue.

We prepared highly cellularised 3D-tissue constructs designed to repair large, complex abdominal wall defects using a poly (lactic acid) (PLLA)-collagen scaffolds in vitro using a flow perfusion bioreactor. The PLLA-collagen scaffolds had a unique structure consisting of a collagen sponge formed within the pores of a mechanically stable knitted mesh of PLLA.

The effect of the flow perfusion bioreactor culturing conditions was investigated in vitro on scaffolds seeded with dermal fibroblasts. The cultured constructs were subsequently studied subcutaneously (SC). The results of in vitro studies demonstrated that the perfusion system facilitated increased cell proliferation and homogenous distribution in the PLLA-collagen scaffolds compared to static conditions.

A highly cellularised 3D-tissue construct was formed by 7 days incubation under perfusion conditions, with increased cellularity by the 28 day time point. The SC experiments demonstrated that implanting constructs with high cellularity resulted in exceptional cell stabilisation, with the survival of implanted cells and expression of the phenotypically-relevant extracellular matrix proteins collagen types I and III, studied by fluorescence in situ hybridisation (FISH) and immunohistochemistry.

The results of the study indicate that highly cellularised 3D-tissue constructs matured in vitro before implantation are requisite to the dramatic cell ingrowths observed in vivo. The high fibroblast number served as a "feeder layer" in the tissue regeneration process. The results also indicate that the PLLA-collagen scaffold architecture and especially pore interconnectivity offer an important platform for abdominal wall engineering applications.

The implantation of this novel porous PPLA-collagen scaffold seeded with dermal fibroblasts following in vitro maturation using a flow perfusion bioreactor system suggests a significant advance over current state-of-the-art procedures for the reconstruction of large, complex abdominal wall tissue defects.

by: Nick Rhodes

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