A two-sided coculture model as a simple tool to track and study cellular events mimicking osteoporosis

摘要

Osteoporosis affects nearly 30% of the female population annually at postmenopausal stage worldwide and adds a huge socioeconomic burden to the aging population in the western world. Clinically, bisphosphonates are the first line of treatment of osteoporosis. However, due to ineffective methods of targeting and delivery, it has been observed that low patient compliance is attributed to unrelated non-specific tissue effects such as gastrointestinal irritation, musculoskeletal pain and other severe effects such as osteonecrosis of the jaw and atypical stress fractures of the long bones. In this study, we demonstrate through a novel two-sided in vitro co-culture model that chemically linking bisphosphonates to biodegradable polymers like hyaluronic acid enhances the specificity of bisphosphonates by targeting osteoclasts and reduces off-target side effects thereby offering better delivery strategies at significantly lower dosages. We have in addition developed a two-sided coculture model with osteoclasts and osteoblasts to study the role and targeting ability of such soluble materials and drugs on surface remodelling events such as resorption and remineralization. EXPERIMENTAL METHODS RAW 264.7 cells or Peripheral Blood Monocytes (PBMC's) were differentiated on one side of bone discs (Bone Slices, Denmark) according to published procedures for 21 days. MC3T3-E1 cells or Saos cells were cultured on the opposite side of the bone disc and differentiated using previously described procedures. After 21 days of differentiation, the cells washed with 1X PBS and cultured for 24 hours with different doses of neutralized HA-BP in a concentration dependent manner. At 24 hrs, the cells were washed with 1X PBS, fixed with 4% PFA and cells were TRAcP stained for osteoclastic activity. Scanning Electron Microscopy (SEM) was performed post-processing the discs to observe resorption pits.. RESULTS AND DISCUSSION After the differentiation period of 21 days, the bone discs were processed using Alkaline phosphatase (ALP) and Alizarin Red staining to identify the extent of mineralization and SEM and TRAP staining to identify resorption. Electron microscopy revealed large number of resorption pits with size range between 10um to 100um. TRAcP staining for osteoclast activity revealed numerous multinucleated cells. Cells with three or more nuclei were classified as osteoclasts using Image J for analysis. The BP treatment on the 2-sided culture revealed a controlled reduction in the number of osteoclasts compared to discs with osteoclasts alone. The quality of mineralization by osteoblasts and the resorptive activity of the osteoclasts were unaffected in all the conditions, verified by electron microscopy and analysis of the metabolic activity of the cells as well as through ALP measurements. Here we demonstrate the potency of Pamidronate linked to low molecular weight hyaluronic acid against murine and human osteoclasts both in single and two-sided cultures. Osteoblasts and osteoclasts together in two-sided culture result in fewer mature osteoclasts that could be attributed to cellular cross talk via soluble factors. In comparison with the conventional co-culture model, we observed that physically separating the osteoblasts and osteoclasts and providing access to the same soluble factors as cytokines or mineralizing agents was not significantly different, thus suggesting that other modes of communication are also in play when drugs or soluble factors interact with two or more different cell types on the same biomaterial. CONCLUSION These results encourage the possibility of applying new delivery mechanisms as well as a novel in vitro method to test the effect of soluble drugs like bisphosphonates and biomaterials on bone related cells. This opens up the possibility of testing drugs at a lower dose and provides more useful information on the possible interaction of the drug with the behaviour of two different cell types, therefore moving a step closer to the in vivo scenario.