Objectives

The biomembrane (induced membrane) formed around polymethylmethacrylate (PMMA) spacers has value in clinical applications for bone defect reconstruction. Few studies have evaluated its cellular, molecular or stem cell features. Our objective was to characterise induced membrane morphology, molecular features and osteogenic stem cell characteristics.

Introduction: The anterior cruciate ligament (ACL) is the most frequently damaged ligament in the knee joint. The patella tendon autograft is the current replacement of choice, however autografts are not always available and grafting often leads to donor site morbidity. Allogeneic implants may cause an adverse immunological reaction [1] The aim of this study was to develop an acellular tendon scaffold with the mechanical and biochemical properties of tissue which could be rapidly recellularised for use in tissue engineering of the anterior cruciate ligament.

Materials and Methods: Porcine patella tendons were dissected less than 24 hours after slaughter and washed in PBS. The tendons were decellularised using 0.1% (w/ v) SDS for 24 hours. Decellularisation was assessed by haematoxylin and eosin staining and light microscopy. The glycosaminoglycan and hydroxyproline (measure of collagen) content of the scaffold were also assessed quantitatively following decellularisation. Following decellularisation the scaffolds were subject to various levels of ultrasonication in order to modify the acellular scaffold prior to reseeding in an attempt to achieve recellularisation of the scaffold. Denaturation of the collagen within the scaffold following ultrasonication was assessed using the ƒÑ-chymotrypsin assay. Decellularised and ultrasonicated scaffolds were subject to uniaxial tensile loading to failure in a Howden tensile testing machine. The sonicated scaffolds were reseeded with human tenocytes (1x105 cells.cm2) and cultured in 5% CO2 in air at 37°C for three weeks. One scaffold was removed every seven days and either fixed in 10% neutral buffered formalin prior to dehydration and H& E staining or was stained with Live/Dead stain (Molecular Probes) and observed using confocal microscopy.

Results: Porcine patella tendons were successfully decellularised using 0.1% (w/v) SDS. Following decellularisation there was no change in the biochemical composition of the scaffold. Ultrasonication of the scaffold at 360W was shown to open up spaces between collagen bundles without damaging the collagen matrix and this was confirmed with the Ą-chymotrypsin assay. Following decellularisation and ultrasonication there was no change in the ultimate force (N) needed to break the tendon scaffold. When cells were seeded onto the sonicated scaffold, the cells were shown to penetrate to the centre of the scaffold within just 3 weeks of culture. Following staining with Live/Dead stain it was shown that after three weeks in static culture approximately 50% of the cells in the centre of the scaffold were viable. In comparison the cells cultured on the acellular non-sonicated scaffold remained on the surface of the scaffold and did not penetrate the matrix during this culture period.

Conclusion: An acellular scaffold with excellent biochemical and mechanical properties has been developed which can be recellularised in an important first step towards tissue engineering of the anterior cruciate ligament. Future work will investigate culture of the reseeded scaffold under appropriate physical stimulation with a view to maintaining tissue homeostasis and increasing cell viability.

Ultra high molecular weight polyethylene (UHMWPE) wear debris induced osteolysis is a major cause of long term failure of total hip replacements. Particles in the 0.1–1.0_m size range are believed to have greater osteolytic potential than larger wear debris. Crosslinked polyethylenes have been shown to have improved wear resistance compared to non-crosslinked materials on smooth counterfaces, however wear debris from cross-linked UHMWPE has been shown to be smaller than that produced from non-crosslinked materials. The aim of this study was to compare the wear, wear debris and biological activity of non-crosslinked and crosslinked polyethylenes when worn against smooth and scratched counterfaces.

Materials and Methods: Test pins were machined from non-crosslinked GUR1050 and GUR1050 crosslinked with either 5 or 10Mrad of gamma irradiation. Sterile endotoxin free clinically relevant wear debris was generated using a bi-directional pin-on-plate test rig. Tests were performed on scratched (Rp=1.0mm) or smooth (Ra=0.02mm) counterfaces. Particles were cultured with murine macrophages at particle volume (mm³): cell number ratios of 50:1,10:1,1:1 and 0.1:1. The levels of TNF-a produced were determined by ELISA following 0,2,4,6,8,24 and 48 hours of culture.

Results: On both smooth and scratched counterfaces crosslinked polyethylene had lower wear than non-crosslinked polyethylene. Determination of the volume distribution of the wear debris demonstrated a greater percentage of wear debris in the submicrometre size range from crosslinked material when worn on scratched counterfaces. Analysis of the debris when worn on smooth counterfaces showed a further reduction in size of debris with particles observed below 100nm in size which reduced the percentage of debris in the sub-micrometre size range for both materials. Crosslinked material worn against scratched counterfaces generated wear debris that was able to stimulate macrophages to produce significant levels of TNF-a after just six hours of co-culture at the highest volumetric concentration and after 24 hours at lower volumetric concentrations. The non-crosslinked material was able to stimulate macrophages only after 24 hours at the highest volumetric concentration. There were no differences between the biological activity of the particles from the 3 materials articulating on the smooth counterfaces they were only able to stimulate significant TNF-a release following 24 hour with the highest volumetric concentration.

Discussion: Although wear resistance is increased by cross-linking on both smooth and scratched counterfaces, when worn against a scratched counterface crosslinked polyethylene generated a greater percentage of debris in the 0.1–1.0mm size range than non-crosslinked polyethylene and this led to an increase in biological activity. However when worn against smooth counterfaces the production of nanometre size wear particles by both materials reduced the volume of debris in the 0.1–1.0mm size range which in turn lead to a lower biological activity.