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Human Osteoblasts' Response to Biomaterials for Subchondral Bone Regeneration in Standard and Aggressive Environments.

ABSTRACT: Osteochondral lesions, when not properly treated, may evolve into osteoarthritis (OA), especially in the elderly population, where altered joint function and quality are usual. To date, a collagen/collagen-magnesium-hydroxyapatite (Col/Col-Mg-HAp) scaffold (OC) has demonstrated good clinical results, although suboptimal subchondral bone regeneration still limits its efficacy. This study was aimed at evaluating the in vitro osteogenic potential of this scaffold, functionalized with two different strategies: the addition of Bone Morphogenetic Protein-2 (BMP-2) and the incorporation of strontium (Sr)-ion-enriched amorphous calcium phosphate (Sr-ACP) granules. Human osteoblasts were seeded on the functionalized scaffolds (OC+BMP-2 and OC+Sr-ACP, compared to OC) under stress conditions reproduced with the addition of H₂O₂ to the culture system, as well as in normal conditions, and evaluated in terms of morphology, metabolic activity, gene expression, and matrix synthesis. The OC+BMP-2 scaffold supported a better osteoblast morphology and stimulated scaffold colonization, cell activity, and extracellular matrix secretion, especially in the stressed culture environment but also in normal culture conditions, with increased expression of genes related to osteoblast differentiation. In conclusion, the incorporation of BMP-2 into the Col/Col-Mg-HAp scaffold also represents an improvement of the osteochondral scaffold in more challenging conditions, supporting further preclinical studies to optimize it for use in clinical practice.

SUBMITTER: Pagani S

PROVIDER: S-EPMC10573262 | biostudies-literature | 2023 Sep

REPOSITORIES: biostudies-literature

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Human Osteoblasts' Response to Biomaterials for Subchondral Bone Regeneration in Standard and Aggressive Environments.

Pagani Stefania S Salerno Manuela M Filardo Giuseppe G Locs Janis J van Osch Gerjo J V M GJVM Vecstaudza Jana J Dolcini Laura L Borsari Veronica V Fini Milena M Giavaresi Gianluca G Columbaro Marta M

International journal of molecular sciences 20230929 19

Osteochondral lesions, when not properly treated, may evolve into osteoarthritis (OA), especially in the elderly population, where altered joint function and quality are usual. To date, a collagen/collagen-magnesium-hydroxyapatite (Col/Col-Mg-HAp) scaffold (OC) has demonstrated good clinical results, although suboptimal subchondral bone regeneration still limits its efficacy. This study was aimed at evaluating the in vitro osteogenic potential of this scaffold, functionalized with two different ...[more]

PMID: 37834212

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Project description:Insulin-like growth factor (IGF)-1 is a key factor in bone homeostasis and could be involved in bone tissue sclerosis as observed in osteoarthritis (OA). Here, we compare the key signaling pathways triggered in response to IGF-1 stimulation between normal and OA osteoblasts (Obs). Primary Obs were prepared from the subchondral bone of tibial plateaus of OA patients undergoing knee replacement or from normal individuals at autopsy. Phenotypic characterization of Obs was evaluated with alkaline phosphatase and osteocalcin release. The effect of IGF-1 on cell proliferation, alkaline phosphatase and collagen synthesis was evaluated in the presence or not of 50 ng/ml IGF-1, whereas signaling was studied with proteins separated by SDS-PAGE before western blot analysis. We also used immunoprecipitation followed by western blot analysis to detect interactions between key IGF-1 signaling elements. IGF-1 receptor (IGF-1R), Shc, Grb2, insulin receptor substrate (IRS)-1, and p42/44 mitogen-activated protein kinase (MAPK) levels were similar in normal and OA Obs in the presence or absence of IGF-1. After IGF-1 stimulation, the phosphorylation of IGF-1R in normal and OA Obs was similar; however, the phosphorylation of IRS-1 was reduced in OA Ob. In addition, the PI3K pathway was activated similarly in normal and OA Obs while that for p42/44 MAPK was higher in OA Obs compared to normal. p42/44 MAPK can be triggered via an IRS-1/Syp or Grb2/Shc interaction. Interestingly, Syp was poorly phosphorylated under basal conditions in normal Obs and was rapidly phosphorylated upon IGF-1 stimulation, yet Syp showed a poor interaction with IRS-1. In contrast, Syp was highly phosphorylated in OA Obs and its interaction with IRS-1 was very strong initially, yet rapidly dropped with IGF-1 treatments. The interaction of Grb2 with IRS-1 progressively increased in response to IGF-1 in OA Obs whereas this was absent in normal Ob. IGF-1 stimulation altered alkaline phosphatase in Ob, an effect reduced in the presence of PD98059, an inhibitor of p42/44 MAPK signaling, whereas neither IGF-1 nor PD98059 had any significant effect on collagen synthesis. In contrast, cell proliferation was higher in OA Obs compared to normal under basal conditions, and IGF-1 stimulated more cell proliferation in OA Obs than in normal Ob, an effect totally dependent on p42/44 MAPK activiy. The altered response of OA Obs to IGF-1 may be due to abnormal IGF-1 signaling in these cells. This is mostly linked with abnormal IRS-1/Syp and IRS-1/Grb2 interaction in these cells.

Project description:BackgroundOsteoarthritis (OA) is the most common human joint disease. Recent studies suggest that an abnormal subchondral bone metabolism is intimately involved in the genesis of this disease. Bone remodelling is tightly regulated by a molecular triad composed of OPG/RANK/RANKL. RANKL exists as 3 isoforms: RANKL1, 2, and 3. RANKL1 and 2 enhance osteoclastogenesis whereas RANKL3 inhibits this phenomenon. We previously reported that human OA subchondral bone osteoblasts can be discriminated into two subgroups according to their level of PGE2 [low (L) or high (H)]. Moreover, we also showed that L-OA osteoblasts express higher levels of total RANKL compared to H-OA osteoblasts. In this study, we investigated the level of membranous RANKL, comparing L- and H-OA subchondral bone osteoblasts, as well as its modulation by osteotropic factors. The impact of the modulation of RANKL1 and 3 on the membranous RANKL level was also studied.MethodsGene expression was determined using real-time PCR for RANKL1 and semi-quantitative PCR for RANKL3. Membranous RANKL was measured by flow cytometry. The modulation of membranous RANKL and RANKL isoforms was monitored on the L- and H-OA osteoblasts and also following treatment with osteotropic factors, including vitamin D3 (50 nM), IL-1beta (100 pg/ml), TNF-alpha (5 ng/ml), PGE2 (500 nM), PTH (100 nM), IL-6 (10 ng/ml) and IL-17 (10 ng/ml).ResultsMembranous RANKL levels were significantly increased in L-OA osteoblasts compared to normal (p<0.01) and H-OA (p<0.05). The gene expression level of the RANKL1 profile was reminiscent of the membranous RANKL level. Although RANKL3 gene expression was lower on the H-OA osteoblasts than on normal and L-OA osteoblasts (p<0.03), the overall outcome favoured RANKL1. Treatment with the tested factors showed a significant increase in membranous RANKL on the L-OA osteoblasts, with the exception of PTH and IL-17. Interestingly in this subpopulation, the RANKL3 gene expression level was significantly increased upon PTH and IL-17 treatment. No effect of the tested osteotropic factors was found on the H-OA.ConclusionOur findings showed that the normal, L- and H-OA subchondral bone osteoblasts differentially express membranous RANKL and RANKL isoforms, and that treatment with osteotropic factors generally favours increased membranous localization of RANKL on L-OA compared to H-OA osteoblasts. This phenomenon appears to take place through differential modulation of each RANKL isoform.

Dataset Information

Human Osteoblasts' Response to Biomaterials for Subchondral Bone Regeneration in Standard and Aggressive Environments.

Publications

Human Osteoblasts' Response to Biomaterials for Subchondral Bone Regeneration in Standard and Aggressive Environments.

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