| Objectives:Within the general aim of finding affordable and sustainable regenerative solutions for damaged and diseased tissues and organs, significant efforts have been invested in developing synthetic alternatives to natural bone grafts, such as autografts. Calcium phosphate (CaP) ceramics are among widely used synthetic bone graft substitutes, but their mechanical properties and bone regenerative capacity are still outperformed by their natural counterparts. While retaining their synthetic character, various strategies have been considered to improve the bioactivity of bone graft substitutes, such as variationsin chemical composition and surface topography. However, CaP ceramics are relatively complex materials, with an intertwined set of physico-chemical properties. This makes it difficult to modify one property without thereby affecting one or more of the others. As a result, correlating any type of biological response to one or a combination of properties is difficult, and yet this is a very important step in the process of designing new bone graft substitutes with functionality equal to the autograft. In order to improve the existing synthetic bone graft substitutes, it is imperative to understand the effects of their individual properties on a biological response, and to find a way to combine the desired properties into new, improved functional biomaterials. To this end, a composite material consisting of poly(lactic acid) (PLA) and nanometer-sized HA powder with engineered surface features at the micrometer scale was used. And the independent effects of the chemical composition and surface microstructure of a poly(lactic acid)/hydroxyapatite (PLA/HA) composite material on the proliferation and osteogenic differentiation of clinically relevant bone marrow-derived human mesenchymal stromal cells (hMSCs) were assessed. While the molecular weight of the polymer and presence/absence of the ceramic phase were used as the chemical variables, a soft embossing technique was used to pattern the surfaces of all materials with either pits or pillars with identical microscale dimensions.Methods:Two types of PLA/HA composite materials were prepared, one using low-molecular-weight poly (D, L-lactic acid) (L-PLA), and one using high molecular weight PLA (H-PLA). Control materials made of L-PLA or H-PLA without HA were produced using the same procedure. Pulsed femtosecond laser micromachining was used to produce a stainless steel master containing the pits topographies used in this study. It's negative replica (thus with pillars) was made and used to produce a second replica (with pits). Both replicas were subsequently used as molds to pattern surfaces of composite and polymer pelletsby hot embossing. Samples embossed with flat surface mold without topographyserved as controls. Then the passage 3 hMSCs were cultured on the different materials with different topographies. For the analysis of cell morphology,4000 cells were seeded on each sample in basic medium (BM). After 3 days of culture, cell morphology was observed with scanning electron microscopy (SEM) and fluorescence microscopy. The analysis of the cell shape parameters, i.e. cell area, cell perimeter and minor axis length was performed using CellProfiler. For the assessment of proliferation, osteogenic differentiation and gene expression of cells, and for the analysis of Ca2+and Pi concentration and material surface changes during culture,10,000 hMSCs were seeded on each sample and incubated in either BM or osteogenic medium (OM). Cell proliferation was assessed after 3,7 and 14 days of culture by measuring total DNA amount. For osteogenic differentiation, Alkaline phosphatase (ALP) activity was determined after 7 and 14 days of culture. To evaluate the influence of surface topography and chemical composition of the samples on the expression of the markers of osteogenic differentiation at the mRNA level, hMSCs were cultured for 7 and 14 days. Total RNA of the cells was extracted and first strand cDNA was synthesized. Then, a series of bone-related genes, bone morphogenetic protein 2 (BMP-2), runt-related transcription factor 2 (Runx2), osteopontin (OP), ALP and collagen type 1 (Col-1) were detected using quantitative real-time PCR. To analyse the effect of Ca2+ and Pi on bone osteogenic differentiation, Ca2+and Pi content of the medium was determined after 3,7 and 14 days of culture. To evaluate the surface topography change, the samples were observed using SEM and energy dispersive X-ray spectroscopy(EDS) at day 14.Results:(1) All the surface of the four materials L-PLA/HA, H-PLA/HA, L-PLA and H-PLA were successfully patterned with either pits or pillars. Elemental mapping of calcium and phosphorous demonstrated a homogenous distribution of the ceramic phase in the composites, independent of the molecular weight of PLA. (2) While cell morphology was affected by both the presence and availability of HA and by the surface microstructure, the effect of the latter parameter was more pronounced. (3) The chemistry, and more specifically, the presence of CaP, had a positive effect on cell proliferation, whereas the effect of surface topography was negligible. (4) The osteogenic differentiation of hMSCs, and in particular the expression of BMP-2 and OP were significantly enhanced when cells were cultured on the composite based on low-molecular-weight PLA, as compared to the high-molecular-weight PLA-based composite and the two pure polymers. The OP expression on the low-molecular-weight PLA-based composite was further enhanced when the surface was patterned with pits. The addition of dexamethasone strongly suppressed the expression of BMP-2 and OP at both 7 and 14 days, while it significantly increased the expression of ALP at both time points as compared to BM. Furthermore, the effect of biomaterials properties, with an emphasis on chemistry, seemed less pronounced as compared to the culture in BM. Taken together, within this experimental set up, the individual effect of the chemistry, and in particular of the presence of CaP, was more pronounced than the individual effect of the surface microstructure, although their combined effects were, in some cases, synergistic. (5) Ionic concentration analysis indicated that cell culture medium (both BM and OM) containing L-PLA/HA contained significantly lower Ca2+ and Pi concentrations as compared to the other three chemistries, independent of the topography. (6) After 14 days of cell culture, an extensive number of submicronpores were observed in L-PLA/HA on both flat and patterned surfaces, whileno such pores were observed on H-PLA/HA surface. Both P/C and Ca/C ratios increased for both L-PLA/HA and H-PLA/HA during 14-day cell culture, while the Ca/P remained fairly constant.Conclusions:This study demonstrated that combining ceramic/polymer composite materials based on a polymer with different molecular weights, with soft embossing is a successful way to decouple the individual effects of chemical composition and surface microstructure on the behavior of hMSCs. While surface micropatterns had a strong effect on cell attachment and morphology, the effect of chemistry, i.e. (the extent of) the presence of CaP had a more pronounced effect on cell proliferation and osteogenic differentiation, in particular in the case of calcium-responsive genes. The approach presented here opens new routes to study the interactions of biomaterials with the biological environment in greater depths, which can serve as a starting point for developing biomaterials with improved bioactivity. |
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