Correction: Human decellularized bone scaffolds from aged donors show improved osteoinductive capacity compared to young donor bone.

[This corrects the article DOI: 10.1371/journal.pone.0177416.].

Human decellularized bone scaffolds from aged donors show improved osteoinductive capacity compared to young donor bone.

To improve the safe use of allograft bone, decellularization techniques may be utilized to produce acellular scaffolds. Such scaffolds should retain their innate biological and biomechanical capacity and support mesenchymal stem cell (MSC) osteogenic differentiation. However, as allograft bone is derived from a wide age-range, this study aimed to determine whether donor age impacts on the ability an osteoinductive, acellular scaffold produced from human bone to promote the osteogenic differentiation of bone marrow MSCs (BM-MSC). BM-MSCs from young and old donors were seeded on acellular bone cubes from young and old donors undergoing osteoarthritis related hip surgery. All combinations resulted in increased osteogenic gene expression, and alkaline phosphatase (ALP) enzyme activity, however BM-MSCs cultured on old donor bone displayed the largest increases. BM-MSCs cultured in old donor bone conditioned media also displayed higher osteogenic gene expression and ALP activity than those exposed to young donor bone conditioned media. ELISA and Luminex analysis of conditioned media demonstrated similar levels of bioactive factors between age groups; however, IGF binding protein 1 (IGFBP1) concentration was significantly higher in young donor samples. Additionally, structural analysis of old donor bone indicated an increased porosity compared to young donor bone. These results demonstrate the ability of a decellularized scaffold produced from young and old donors to support osteogenic differentiation of cells from young and old donors. Significantly, the older donor bone produced greater osteogenic differentiation which may be related to reduced IGFBP1 bioavailability and increased porosity, potentially explaining the excellent clinical results seen with the use of allograft from aged donors.

Polyurethane scaffolds seeded with CD34(+) cells maintain early stem cells whilst also facilitating prolonged egress of haematopoietic progenitors.

We describe a 3D erythroid culture system that utilises a porous polyurethane (PU) scaffold to mimic the compartmentalisation found in the bone marrow. PU scaffolds seeded with peripheral blood CD34(+) cells exhibit a remarkable reproducibility of egress, with an increased output when directly compared to human bone scaffolds over 28 days. Immunofluorescence demonstrated the persistence of CD34(+) cells within the scaffolds for the entirety of the culture. To characterise scaffold outputs, we designed a flow cytometry panel that utilises surface marker expression observed in standard 2D erythroid and megakaryocyte cultures. This showed that the egress population is comprised of haematopoietic progenitor cells (CD36(+)GPA(-/low)). Control cultures conducted in parallel but in the absence of a scaffold were also generally maintained for the longevity of the culture albeit with a higher level of cell death. The harvested scaffold egress can also be expanded and differentiated to the reticulocyte stage. In summary, PU scaffolds can behave as a subtractive compartmentalised culture system retaining and allowing maintenance of the seeded "CD34(+) cell" population despite this population decreasing in amount as the culture progresses, whilst also facilitating egress of increasingly differentiated cells.

Development of an improved bone washing and demineralisation process to produce large demineralised human cancellous bone sponges.

Shaped demineralised bone matrices (DBM) made from cancellous bone have important uses in orthopaedic and dental procedures, where the properties of the material allow its insertion into confined defects, therefore acting as a void filler and scaffold onto which new bone can form. The sponges are often small in size, <1.0 cm(3). In this study, we report on an improved bone washing and demineralisation process that allows production of larger DBM sponges (3.375 or 8.0 cm(3)) from deceased donor bone. These sponges were taken through a series of warm water washes, some with sonication, centrifugation, 100 % ethanol and two decontamination chemical washes and optimally demineralised using 0.5 N hydrochloric acid under vacuum. Demineralisation was confirmed by quantitative measurement of calcium and qualitatively by compression. Protein and DNA removal was also determined. The DBM sponges were freeze dried before terminal sterilisation with a target dose of 25 kGy gamma irradiation whilst frozen. Samples of the sponges were examined histologically for calcium, collagen and the presence of cells. The data indicated lack of cells, absence of bone marrow and a maximum of 1.5 % residual calcium.

Optimized demineralization of human cancellous bone by application of a vacuum.

Human demineralized bone matrix derived from cortical bone is used by surgeons due to its ability to promote bone formation. There is also a need for shaped demineralized bone matrices made from cancellous bone, where the properties of the material allow its insertion into defects, therefore acting as a void filler and scaffold onto which new bone can form. In this study, we report that demineralized bone sponges were prepared by dissecting and cutting knee bone into cancellous bone cubes of 1 cm(3) . These cubes were then taken through a series of warm water washes, some with sonication, centrifugation, and two decontamination chemical washes. The cubes were optimally demineralized into sponges with 0.5N hydrochloric acid under vacuum with constant pH measurement. Demineralization was confirmed by quantitative measurement of calcium and qualitatively by compression. The sponges were freeze dried before terminal sterilisation with a target dose of 25 kGy gamma radiation whilst frozen. Samples of the sponges were histologically examined for calcium and collagen and also tested for osteoinductivity. Data showed well defined collagen staining in the sponges, with little residual calcium. Sponges from two out of three donors demonstrated osteoinductivity when implanted into the muscle of an athymic mouse.