Influence of Bone Substitutes on Mesenchymal Stromal Cells in an Inflammatory Microenvironment.

Bone regeneration is driven by mesenchymal stromal cells (MSCs) via their interactions with immune cells, such as macrophages (MPs). Bone substitutes, e.g., bi-calcium phosphates (BCPs), are commonly used to treat bone defects. However, little research has focused on MSC responses to BCPs in the context of inflammation. The objective of this study was to investigate whether BCPs influence MSC responses and MSC-MP interactions, at the gene and protein levels, in an inflammatory microenvironment. In setup A, human bone marrow MSCs combined with two different BCP granules (BCP 60/40 or BCP 20/80) were cultured with or without cytokine stimulation (IL1ß + TNFα) to mimic acute inflammation. In setup B, U937 cell-line-derived MPs were introduced via transwell cocultures to setup A. Monolayer MSCs with and without cytokine stimulation served as controls. After 72 h, the expressions of genes related to osteogenesis, healing, inflammation and remodeling were assessed in the MSCs via quantitative polymerase chain reactions. Additionally, MSC-secreted cytokines related to healing, inflammation and chemotaxis were assessed via multiplex immunoassays. Overall, the results indicate that, under both inflammatory and non-inflammatory conditions, the BCP granules significantly regulated the MSC gene expressions towards a pro-healing genotype but had relatively little effect on the MSC secretory profiles. In the presence of the MPs (coculture), the BCPs positively regulated both the gene expression and cytokine secretion of the MSCs. Overall, similar trends in MSC responses were observed with BCP 60/40 and BCP 20/80. In summary, within the limits of in vitro models, these findings suggest that the presence of BCP granules at a surgical site may not necessarily have a detrimental effect on MSC-mediated wound healing, even in the event of inflammation.

Influence of Type 2 Diabetes on Prevalence of Key Periodontal Pathogens, Salivary Matrix Metalloproteinases, and Bone Remodeling Markers in Sudanese Adults with and without Chronic Periodontitis.

This study compared the influence of type 2 diabetes on the occurrence of six periodontal pathogens in plaque samples of patients with and without chronic periodontitis. Levels of salivary MMP-8, MMP-9, RANKL, and OPG were also investigated. The study enrolled 31 patients with type 2 diabetes and chronic periodontitis (DM + CP), 29 with chronic periodontitis (CP), and 20 with type 2 diabetes (DM). Questionnaire-guided interviews were conducted and plaque index, bleeding on probing, and pocket depth were recorded. Polymerase chain reaction (PCR) was utilized to determine the prevalence of the bacteria. The levels of salivary molecules were determined by enzyme immunosorbent assay (ELISA). The CP group had the highest prevalence of P. gingivalis (81.5%), followed by the DM + CP (59.3%) and DM (55.0%) groups (P > 0.05). Similar trends were observed for P. intermedia and T. denticola. The prevalence of T. forsythia was 100% in both periodontitis groups compared to 90% in the DM group. There were no significant differences between the groups regarding the concentrations of MMP-8, MMP-9, or OPG. RANKL concentrations were below the detection limit. Our data show that type 2 diabetes has no significant influence on the prevalence of the investigated periodontal pathogens, or the levels of salivary MMP-8, MMP-9, and OPG.

Biodegradable polymer scaffolds loaded with low-dose BMP-2 stimulate periodontal ligament cell differentiation.

Poly(L-lactide)-co-(epsilon-caprolactone) [poly(LLA-co-CL)] and poly(L-lactide)-co-(1,5-dioxepan-2-one) [poly(LLA-co-DXO)] are being considered candidate scaffolds for bone tissue engineering. We evaluated the bioactive potential of poly(LLA-co-CL) and poly(LLA-co-DXO) scaffolds loaded with low-dose bone morphogenetic protein-2 (BMP-2). Periodontal ligament (PDL) cells were cultured onto the various scaffolds loaded with 1 µg BMP-2 or without BMP-2 (control). Cell viability, attachment, and proliferation were determined using a methylthiazol tetrazolium (MTT) colorimetric assay at day 1, 3, and 7. Scanning electron microscopy was used to analyze cell morphology at day 7. Cell differentiation was evaluated assaying alkaline phosphatase (ALP) activity at day 7, 14, and 21. Real-time PCR was used to evaluate the mRNA expression of periostin, ALP, type I collagen, bone sialoprotein and BMP-2. A commercially available enzyme-linked immunosorbent assay was used to assess BMP-2 production. Surface analysis disclosed excellent cell attachment, spread, and penetration into the porous scaffolds. The MTT assay indicated that scaffolds loaded with low concentration of BMP-2 did not influence the viability of cells. Cells grown on the modified scaffolds expressed higher levels of osteogenic markers than the nonmodified scaffolds (p<0.05). Poly(LLA-co-CL) and poly(LLA-co-DXO) scaffolds loaded with low-dose BMP-2 exhibited a significant effect stimulating PDL differentiation suggesting a continued evaluation in relevant in vivo models.

Polyester copolymer scaffolds enhance expression of bone markers in osteoblast-like cells.

In tissue engineering, the resorbable aliphatic polyester poly(L-lactide) (PLLA) is used as scaffolds in bone regeneration. Copolymers of poly(L-lactide)-co-(epsilon-caprolactone) [poly(LLA-co-CL)] and poly(L-lactide)-co-(1,5-dioxepan-2-one) [poly(LLA-co-DXO)], with superior mechanical properties to PLLA, have been developed to be used as scaffolds, but the influence on the osteogenic potential is unclear. This in vitro study of test scaffolds of poly(LLA-co-CL) and poly(LLA-co-DXO) using PLLA scaffolds as a control demonstrates the attachment and proliferation of human osteoblast-like cells (HOB) as measured by SEM and a methylthiazol tetrazolium (MTT) colorimetric assay, and the progression of HOB osteogenesis for up to 3 weeks; expressed as synthesis of the osteoblast differentiation markers: collagen type 1 (Col 1), alkaline phosphatase, bone sialoprotein, osteocalcin (OC), osteopontin and runt related gene 2 (Runx2). Surface analysis disclosed excellent surface attachment, spread and penetration of the cells into the pores of the test scaffolds compared to the PLLA. MTT results indicated that test scaffolds enhanced the proliferation of HOBs. Cells grown on the test scaffolds demonstrated higher synthesis of Col 1 and OC and also increased bone markers mRNA expression. Compared to scaffolds of PLLA, the poly(LLA-co-CL) and poly(LLA-co-DXO) scaffolds enhanced attachment, proliferation, and expression of osteogenic markers by HOBs in vitro. Therefore, these scaffolds might be appropriate carriers for bone engineering.