Objectives: Autogenous bone grafts are the gold standard for bone augmentation procedures with the ability to release growth factors. These growth factors can be isolated into a "bone-conditioned medium" (BCM). No effort has been made to utilise the growth factors from fresh bone chips in combination with biomaterials to improve bone regeneration. This study aimed to investigate the ability of collagen barrier membranes and DBBM treated with BCM to affect cell behaviour.
Methods: Cortical bone chips were harvested from fresh pig mandibles with a bone scraper and placed into plastic dishes containing serum-free culture medium (5g of bone chips per 10mL of medium) for 24 hours. Natural collagen membranes (Bio-GideTM/®) were incubated with BCM for various times. Membranes were also (i) incubated for 4 hours with recombinant TGF-β1; (ii) exposed to ultraviolet light prior to BCM incubation; (iii) pre-wetted for 15 minutes with phosphate buffer saline (PBS) prior to BCM incubation; or (iv) dried and stored at room temperature for 7 days after BCM incubation. After incubation, the membranes were vigorously washed with PBS. DBBM particles (Bio-OssTM/®) were coated with BCM for 5 minutes prior to cell seeding. Gingival fibroblasts or bone-marrow-derived stromal cells (ST2 cells) were seeded on the collagen membranes and DBBM particles, respectively. Messenger RNA levels of BCM target genes were analysed by qRT-PCR using adrenomedullin (ADM), pentraxin 3 (PTX-3), interleukin 11 (IL-11) and proteoglycan-4 (PRG-4). The morphology and viability of cells seeded onto collagen membranes was evaluated. Further, DBBM with and without a BCM coating was compared in terms of cell recruitment, adhesion, proliferation and qRT-PCR for osteoblast differentiation markers (including Runx2, COL1A2, ALP and OCNAlizarin red stain was used to assess mineralisation. The student‘s t-test was used for analysis.
Results: Incubation of collagen membranes with BCM for at least 1 minute reduced fibroblast ADM and PTX-3 expression, and increased IL-11 and PRG-4 expression. The four different membrane treatments (i–iv) also provoked significant changes in gene expression. Likewise, conditioned medium from demineralised bone chips caused similar changes in gene expression compared to BCM. BCM did not alter the viability or morphology of gingival fibroblasts on collagen membranes. Coating BCM on DBBM particles improved cell migration of ST2 cells and led toa two-fold increase in cell adhesion. No significant increase in cell proliferation was observed, but BCM significantly increased mRNA levels of COL1a2, ALP and OCN at 3 days post-seeding. A three-fold increase in alizarin red staining was observed on DBBM particles that were pre-coated with BCM.
Conclusions: Collagen membranes rapidly adsorb the TGF-β activity of bone chips, and pre-coating DBBM with BCM enhances the osteoconductive properties of DBBM by mediating osteoblast recruitment, attachment and differentiation towards an osteoblast phenotype. These cellular effects of BCM, in combination with biomaterials, might contribute to the overall process of guided bone regeneration. Further animal studies are needed to characterise the added benefit of BCM as an autogenous growth factor for combination therapies.