Molecular Dynamic Simulations for Biopolymers with Biomedical Applications.

Computational modeling (CM) is a versatile scientific methodology used to examine the properties and behavior of complex systems, such as polymeric materials for biomedical bioengineering. CM has emerged as a primary tool for predicting, setting up, and interpreting experimental results. Integrating in silico and in vitro experiments accelerates scientific advancements, yielding quicker results at a reduced cost. While CM is a mature discipline, its use in biomedical engineering for biopolymer materials has only recently gained prominence. In biopolymer biomedical engineering, CM focuses on three key research areas: (A) Computer-aided design (CAD/CAM) utilizes specialized software to design and model biopolymers for various biomedical applications. This technology allows researchers to create precise three-dimensional models of biopolymers, taking into account their chemical, structural, and functional properties. These models can be used to enhance the structure of biopolymers and improve their effectiveness in specific medical applications. (B) Finite element analysis, a computational technique used to analyze and solve problems in engineering and physics. This approach divides the physical domain into small finite elements with simple geometric shapes. This computational technique enables the study and understanding of the mechanical and structural behavior of biopolymers in biomedical environments. (C) Molecular dynamics (MD) simulations involve using advanced computational techniques to study the behavior of biopolymers at the molecular and atomic levels. These simulations are fundamental for better understanding biological processes at the molecular level. Studying the wide-ranging uses of MD simulations in biopolymers involves examining the structural, functional, and evolutionary aspects of biomolecular systems over time. MD simulations solve Newton's equations of motion for all-atom systems, producing spatial trajectories for each atom. This provides valuable insights into properties such as water absorption on biopolymer surfaces and interactions with solid surfaces, which are crucial for assessing biomaterials. This review provides a comprehensive overview of the various applications of MD simulations in biopolymers. Additionally, it highlights the flexibility, robustness, and synergistic relationship between in silico and experimental techniques.

Dynamic structure and composition of bone investigated by nanoscale infrared spectroscopy.

Bone is a highly organized tissue in which each structural level influences the macroscopic and microscopic mechanical behavior. In particular, the quantity, quality, and distribution of the different bone components, i.e. collagen matrix and hydroxyapatite crystals, are associated with bone strength or fragility. Common spectroscopic techniques used to assess bone composition have resolutions limited to the micrometer range. In this study, our aims were two-fold: i) to develop and validate the AFM-IR methodology for skeletal tissues and ii) to apply the methodology to sheep cancellous bone with the objective to obtain novel findings on the composition and structure of trabecular packets.To develop the methodology, we assessed spatial and temporal reproducibility using a known homogeneous material (polymethylmethacrylate, PMMA). We verified that the major peak positions were similar and not shifted when compared to traditional Fourier Transform Infrared imaging (FTIRI). When AFM-IR was applied to sheep cancellous bone, the mineral-to-matrix ratio increased and the acid phosphate substitution ratio decreased as a function of tissue maturity. The resolution of the technique enabled visualization of different stages of the bone maturation process, particularly newly-formed osteoid prior to mineralization. We also observed alternating patterns of IR parameters in line and imaging measurements, suggesting the apposition of layers of alternating structure and / or composition that were not visible with traditional spectroscopic methods. In conclusion, nanoscale IR spectroscopy demonstrates novel compositional and structural changes within trabecular packets in cancellous bone. Based on these results, AFM-IR is a valuable tool to investigate cancellous bone at the nanoscale and, more generally, to analyze small dynamic areas that are invisible to traditional spectroscopic methods.

Phosphorylation regulates the secondary structure and function of dentin phosphoprotein peptides.

Dentin phosphoprotein (DPP) is the most acidic protein in vertebrates and structurally is classified as an intrinsically disordered protein. Functionally, DPP is related to dentin and bone formation, however the specifics of such association remain unknown. Here, we used atomistic molecular dynamics simulations to screen selected binding domains of DPP onto hydroxyapatite (HA), which is one of its important interacting partners. From these results, we selected a functionally relevant peptide, Ace-SSDSSDSSDSSDSSD-NH2 (named P5) and its phosphorylated form (named P5P), for experimental characterization. SAXS experiments indicated that in solution P5 was disordered, possibly in an extended conformation while P5P displayed more compact globular conformations. Circular dichroism and FTIR confirmed that, either in the presence or absence of Ca2+/HA, P5 adopts a random coil structure, whereas its phosphorylated counterpart, P5P, has a more compact arrangement associated with conformations that display ß-sheet and α-helix motifs when bound to HA. In solution, P5 inhibited HA crystal growth, whereas at similar concentrations, P5P stimulated it. These findings suggest that phosphorylation controls the transient formation of secondary and tertiary structure of DPP peptides, and, most likely of DPP itself, which in turn controls HA growth in solution and possibly HA growth in mineralized tissues.

Intrinsically disordered proteins and biomineralization.

In vertebrates and invertebrates, biomineralization is controlled by the cell and the proteins they produce. A large number of these proteins are intrinsically disordered, gaining some secondary structure when they interact with their binding partners. These partners include the component ions of the mineral being deposited, the crystals themselves, the template on which the initial crystals form, and other intrinsically disordered proteins and peptides. This review speculates why intrinsically disordered proteins are so important for biomineralization, providing illustrations from the SIBLING (small integrin binding N-glycosylated) proteins and their peptides. It is concluded that the flexible structure, and the ability of the intrinsically disordered proteins to bind to a multitude of surfaces is crucial, but details on the precise-interactions, energetics and kinetics of binding remain to be determined.

The role of phosphorylation in dentin phosphoprotein peptide absorption to hydroxyapatite surfaces: a molecular dynamics study.

Dentin phosphoprotein (DPP) is a protein expressed mainly in dentin and to a lesser extent in bone. DPP has a disordered structure, rich in glutamic acid, aspartic acid and phosphorylated serine/threonine residues. It has a high capacity for binding to calcium ions and to hydroxyapatite (HA) crystal surfaces. We used molecular dynamics (MD) simulations as a method for virtually screening interactions between DPP motifs and HA. The goal was to determine which motifs are absorbed to HA surfaces. For these simulations, we considered five peptides from the human DPP sequence. All-atom MD simulations were performed using GROMACS, the peptides were oriented parallel to the {100} HA crystal surface, the distance between the HA and the peptide was 3 nm. The system was simulated for 20 ns. Preliminary results show that for the unphosphorylated peptides, the acidic amino acids present an electrostatic attraction where their side chains are oriented towards HA. This attraction, however, is slow to facilitate bulk transport to the crystal surface. On the other hand, the phosphorylated (PP) peptides are rapidly absorbed on the surface of the HA with their centers of mass closer to the HA surface. More importantly, the root mean square fluctuation (RMSF) indicates that the average structures of the phosphorylated peptides are very inflexible and elongate, while that of the unphosphorylated peptides are flexible. Radius of gyration (Rg) analysis showed the compactness of un-phosphorylated peptides is lower than phosphorylated peptides. Phosphorylation of the DPP peptides is necessary for binding to HA surfaces.

Characterization of recombinant human cementum protein 1 (hrCEMP1): primary role in biomineralization.

Cementum protein 1 (CEMP1) has been recently cloned, and in vitro experiments have shown functions as regulator of cementoblast behavior and inducer of differentiation of non-osteogenic cells toward a cementoblastic/osteoblastic phenotype. In this study, we have produced a full-length human recombinant CEMP1 protein in a human gingival fibroblast cell line. The purified protein (hrCEMP1) has a M(r) 50,000. Characterization of hrCEMP1 indicates that its secondary structure is mainly composed of beta-sheet (55%), where random coil and alpha helix conformations correspond to 35% and 10%, respectively. It was found that hrCEMP1 is N-glycosylated, phosphorylated and possesses strong affinity for hydroxyapatite. Even more important, our results show that hrCEMP1 plays a role during the biomineralization process by promoting octacalcium phosphate (OCP) crystal nucleation. These features make CEMP1 a very good candidate for biotechnological applications in order to achieve cementum and/or bone regeneration.

Human Cementum Protein 1 induces expression of bone and cementum proteins by human gingival fibroblasts.

We recently presented evidence showing that a human cementoblastoma-derived protein, named Cementum Protein 1 (CEMP1) may play a role as a local regulator of cementoblast differentiation and cementum-matrix mineralization. This protein was shown to be expressed by cementoblasts and progenitor cells localized in the periodontal ligament. In this study we demonstrate that transfection of CEMP1 into human gingival fibroblasts (HGF) induces mineralization and expression of bone and cementum-matrix proteins. The transfected HGF cells had higher alkaline phosphatase activity and proliferation rate and they expressed genes for alkaline phosphatase, bone sialoprotein, osteocalcin, osteopontin, the transcription factor Runx2/Cbfa1, and cementum attachment protein (CAP). They also produced biological-type hydroxyapatite. These findings indicate that the CEMP1 might participate in differentiation and mineralization of nonosteogenic cells, and that it might have a potential function in cementum and bone formation.