Treatment of bone defects remains a critical challenge in the restoration of form and function of lost osseous tissue. Lipoxin A4 (LXA4), derived from arachidonic acid has been reported to promote resolution of inflammation, minimise alveolar bone loss in chronic inflammation and enhance epithelial wound healing. Polyethylene glycol (PEG)-based hydrogels are three dimensional networks of synthetic polymers developed for the application as scaffolds or as carriers for therapeutic agents. The aims of this study were to determine: 1. the effects of LXA4 on the proliferation and differentiation of fibroblasts and osteoblasts, in vitro. 2. the effect of local application of LXA4 on bone healing of a surgical defect in the rat mandible. 3. the effect of PEG-based hydrogels on the proliferation and differentiation of fibroblasts and osteoblasts, in vitro, and 4. the effect of an injectable BoltornTM hydrogel alone and with LXA4, on bone healing of the surgical defect.
Materials and Methods
Primary periodontal fibroblasts and calvarial osteoblasts were obtained from four-week old Lewis rats. The effect of LXA4 and PEG-based hydrogels on the proliferation and viability of fibroblasts and the immunoexpression of cultured fibroblast and osteoblast markers in vitro was determined and compared with controls at days 1, 3 and 5 post-culture. Additionally, cell attachment of fibroblasts to the hydrogel was determined at similar time intervals. In the LXA4-treated groups, LXA4 (0.5μg, 1μg or 2μg) was delivered to the primary fibroblasts and osteoblasts every 2nd day. In hydrogel treated groups, four types of hydrogels including, polyethylene glycol triblock copolymer (PEG triblock), di-acrylated polyethylene glycol (700PEG), BoltornTM H20 copolymers (BH20) and polyhedral oligomeric silsesquioxane POSS (POSS), were tested. In all groups, fibroblast proliferation was determined using cell counts and viability determined using a live/dead cytotoxicity assay. In addition, fibroblast attachment to the hydrogel surface and cell spreading was examined using confocal microscopy. The immunoexpression of markers for fibroblasts, basic fibroblast growth factor (bFGF) and vimentin (VMT), and for osteoblast, alkaline phosphatase (ALP), osteopontin (OPN) and osteocalcin (OCN) were determined. The concentration of ALP and OCN produced from the osteoblast culture was measured by means of ELISA.
Female Lewis rats, 12-weeks-old, were anaesthetised and a standardised surgical defect, with depth and diameter of 1.0mm, placed on the left buccal aspect of the mandible. Post-operatively, LXA4 (1.0μg or 2.0μg /100gm body weight) was delivered to the site of the defect on every third day by intraoral submucosal injection. In the hydrogel-treated group, the BH20 hydrogel, alone or with LXA4 was injected into the surgical defect. To determine the effect of LXA4 or hydrogels, animals were sacrificed at days 5, 10, 20 and 30 post-treatment. Undecalcified mandibles in controls and LXA4 groups only, were scanned using confocal laser scanning microscopy (CLSM) to assess for topographical changes in the depth of the defect. Undecalcified transverse serial sections were stained for histomorphology using haematoxylin and eosin (H&E) and Masson’s trichrome staining, for tartrate resistant acid phosphatase and for the immunoexpression of ALP, OCN, and OPN. In the LXA4-treated groups, immunoexpression of osteoprotegerin (OPG) and receptor activator of nuclear factor kappa B-ligand (RANKL) were also determined. Comparisons between controls and treatment groups were made by analysis of variance (ANOVA).
In control cultures, a gradual increase in the proliferation and viability of the fibroblasts, and the immunoexpression of fibroblast and osteoblast markers, over the 5-day culture period was observed. At day 3, LXA4 enhanced the proliferation and viability of the fibroblasts. By day 5, LXA4 reduced the bFGF-positive and VMT-positive cell numbers and the bFGF-positive cell numbers in the PBS group were also reduced. However, from day 1 onwards, LXA4 enhanced the immunoexpression of bone markers and the ALP concentration, and increased the OCN concentration in the osteoblast culture by day 5.
During healing of the bone defect, at day 5, the defect contained collagen fibres and remnants of blood. At day 10, and the depth of the defect was reduced compared to the initial depth and the defect contained dense collagenous connective tissue, islands of unmineralised bone matrix and mineralised bone. From day 10 onwards, the immunoexpression of bone matrix proteins had increased, suggestive of healing within the surgical defect. At day 20, the depth of the defect was reduced and the defect contained dense collagenous connective tissue, bone matrix and trabecular bone, and osteoclast numbers had increased. By day 30, the depth of the defect was further reduced and was occluded with mineralised bone and the buccal contour of the bone was restored. Following LXA4 treatment during healing of the defect, at day 10, LXA4 initiated early new bone formation and subsequently decreased the depth of the defect. However, LXA4 treatment reduced the number of ALP-positive cells at day 10 and osteoclast counts at day 20. In the day 20 PBS-treated group, the higher osteoclast counts could be associated with trauma and inflammation created by multiple submucosal injections. By day 30, the defect in the LXA4 and PBS groups was partially occluded with new bone resulting in an increase in the depth of the defect.
In vitro, PEG triblock and 700PEG hydrogels did not affect the proliferation, viability and cell attachment of the fibroblasts and the immunoexpression of fibroblast and osteoblast markers, over the 5-day culture period. At day 5, in osteoblast culture containing 700PEG hydrogel, an increase in the ALP and OCN concentration was observed. Cultures with the POSS showed a decrease in fibroblast proliferation and viability and the immunoexpression of fibroblast markers. POSS hydrogel also reduced the cellular expression of OCN and OCN concentration in the osteoblast culture, signifying that hydrogel was not ideal for in vivo bone healing. Cells cultured with the BH20 hydrogel showed a higher proliferation, viability and cell attachment of fibroblasts and immunoexpression of fibroblast and osteoblast markers over time. In addition, osteoblast culture with the BH20 hydrogel showed an increase in OCN concentration at day 5. The BH20 hydrogel demonstrated a better biocompatibility compared with the other hydrogels tested and was selected for in vivo injection into the bone defect.
The BH20 hydrogel injected, alone or with LXA4, into the defect, did not induce inflammation or inhibit healing of the defect, and had completely degraded by day 30. In the undecalcified tissue section from the day 5 hydrogel group, tearing or loss of soft tissue and hydrogels occurred, leaving artifacts within the defect and may indicate the lack of integration of the local cells into the hydrogel contained within the defect. By day 20 and 30, in the hydrogel groups, there was a delay in the new bone formation and the defect was partially occluded with new bone, resulting in a decrease in the immunoexpression of bone matrix proteins at day 20.
The present study demonstrated that in cell cultures, LXA4 enhanced early proliferation and viability of fibroblasts and the immunoexpression of osteoblast markers by day 5. The study using hydrogels provided a basic understanding on the biocompatibility and development of cells and its effect on bone healing. During bone healing, LXA4 promoted early new bone formation and enhanced the immunoexpression of bone matrix proteins in vivo. However, at day 20, LXA4 reduced osteoclasts numbers and altered the bone healing pattern over time. In this study, the application of a hydrogel loaded with LXA4 is an initial investigation into the development of a new therapeutic agent that could be used in the management of bone healing and bone regeneration.