Natural protein bioinspired materials for regeneration of hard tissues.

The regenerative materials for hard tissues, i.e. tooth (enamel, dentin, and cementum) and bone, require extremely high standards in terms of their mechanical properties, biocompatibility, bioactivity, and multiple-functionality. Among them, the biomedical materials inspired from various natural proteins have attracted increasing research attention. These blueprint proteins include various hard-tissue-related proteins, such as collagen and non-collagenous proteins (e.g. amelogenin, dentin phosphoprotein, bone sialoprotein, and osteopontin), as well as other natural proteins like mussel foot proteins. The current review highlights the structure-function relationship of protein bioinspired biomedical materials (e.g. polymers and polypeptides) and their applications for tooth and bone regeneration. Specifically, the materials bioinspired from salivary acquired pellicle proteins, which have a strong affinity to hydroxyapatite surfaces, are discussed in detail. Finally, the challenges associated with these protein bioinspired materials and their industrialization potentials are discussed.

Advances in biomolecule inspired polymeric material decorated interfaces for biological applications.

With the development of surface modification technology, interface properties have great effects on the interaction between biomedical materials and cells and biomolecules, which significantly affects the biocompatibility and functionality of materials. As an orderly and perfect system, biological organisms in nature effectively integrate all kinds of bio-interfaces with physiological functions, which shed light on the importance of biomolecules in organisms. It gives birth to a bio-inspiration strategy to design and fabricate smart materials with specific functionalities, e.g. osteogenic and chondrocytic induced materials inspired by bone sialoprotein and chondroitin sulfate. Through this mimicking approach, various functional materials were utilized to decorate the interfaces and further optimize the performance of biomedical materials, which would widely expand their applications. In this review, followed by a summary and brief introduction of surface modification methods, we highlight recent advances in the fabrication of functional polymeric materials inspired by a range of biomolecules for decorating interfaces. Then, the other applications of biomolecule inspired materials including tissue engineering, diagnosis and treatment of diseases and physiological function regulation are presented and the future outlook is discussed as well.

Construction of vascularized tissue-engineered bone with a double-cell sheet complex.

A double-cell sheet (DCS) complex composed of an osteogenic cell sheet and a vascular endothelial cell sheet with osteogenesis and blood vessel formation potential was developed in this study. The osteogenic and vascular endothelial cell sheets were obtained after induced culture of rabbit adipose-derived mesenchymal stem cells. The osteogenic cell sheet showed positive alizarin red, von Kossa, and alkaline phosphatase (ALP) staining. The vascular endothelial cell sheet exhibited visible W-P bodies in the cells, the expression of CD31 was positive, and a vascular mesh structure was spontaneously formed in a Matrigel matrix. The subcutaneous transplantation results for four groups of DCS and DCS-coral hydroxyapatite (CHA) complexes, and the CHA scaffold group in nude mice revealed mineralization of collagen fibers and vascularization in each group at 12 weeks, but the degrees of mineralization and vascularization showed differences among groups. The pattern involving endothelial cell sheets covered with osteogenic cell sheets, group B, exhibited the best results. In addition, the degree of mineralization of the DCS-CHA complexes was more mature than those of the same group of DCS complexes and the CHA scaffold, and the capillary number was greater than those of the same group of DCS complexes and the CHA scaffold. Therefore, the CHA scaffold strengthened the osteogenesis and blood vessel formation potential of the DCS complexes. Meanwhile, the DCS complexes also promoted the osteogenesis and blood vessel formation potential of the CHA scaffold. This study will provide a basis for building vascularized tissue-engineered bone for bone defect therapy. STATEMENT OF SIGNIFICANCE: This study developed a double-cell sheet (DCS) complex composed of an osteogenic cell sheet and a vascular endothelial cell sheet with osteogenesis and blood vessel formation potential. Osteogenic and vascular endothelial cell sheets were obtained after induced culture of rabbit adipose-derived mesenchymal stem cells. The DCS complex and DCS-CHA complex exhibited osteogenic and blood vessel formation potential in vivo. CHA enhanced the osteogenesis and blood vessel formation abilities of the DCS complexes in vivo. Meanwhile, the DCS complexes also promoted the osteogenesis and blood vessel formation potential of the CHA scaffold. Group B of the DCS complexes and DCS-CHA complexes exhibited the best osteogenesis and blood vessel formation abilities.

Effect of bone sialoprotein coated three-dimensional printed calcium phosphate scaffolds on primary human osteoblasts.

The combination of the two techniques of rapid prototyping 3D-plotting and bioactive surface functionalization is presented, with emphasis on the in vitro effect of Bone Sialoprotein (BSP) on primary human osteoblasts (hOBs). Our primary objective was to demonstrate the BSP influence on the expression of distinctive osteoblast markers in hOBs. Secondary objectives included examinations of the scaffolds' surface and the stability of BSP-coating as well as investigations of cell viability and proliferation. 3D-plotted calcium phosphate cement (CPC) scaffolds were coated with BSP via physisorption. hOBs were seeded on the coated scaffolds, followed by cell viability measurements, gene expression analysis and visualization. Physisorption is an effective method for BSP-coating. Coating with higher BSP concentrations leads to enhanced BSP release. Two BSP concentrations (50 and 200 µg/mL) were examined in this study. The lower BSP concentration (50 µg/mL) decreased ALP and SPARC expression, whereas the higher BSP concentration (200 µg/mL) did not change gene marker expression. Enhanced cell viability was observed on BSP-coated scaffolds on day 3. hOBs developed a polygonal shape and connected in an intercellular network under BSP influence. Quantitative cell morphology analyses demonstrated for BSP-coated CPCs an enhanced cell area and reduced circularity. The strength of the above-mentioned effects of BSP-coated scaffolds in vivo is unknown, and future work is focusing on bone ingrowth and vascularization in vivo. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 106B: 2565-2575, 2018.

Surface Functionalization of Orthopedic Titanium Implants with Bone Sialoprotein.

Orthopedic implant failure due to aseptic loosening and mechanical instability remains a major problem in total joint replacement. Improving osseointegration at the bone-implant interface may reduce micromotion and loosening. Bone sialoprotein (BSP) has been shown to enhance bone formation when coated onto titanium femoral implants and in rat calvarial defect models. However, the most appropriate method of BSP coating, the necessary level of BSP coating, and the effect of BSP coating on cell behavior remain largely unknown. In this study, BSP was covalently coupled to titanium surfaces via an aminosilane linker (APTES), and its properties were compared to BSP applied to titanium via physisorption and untreated titanium. Cell functions were examined using primary human osteoblasts (hOBs) and L929 mouse fibroblasts. Gene expression of specific bone turnover markers at the RNA level was detected at different intervals. Cell adhesion to titanium surfaces treated with BSP via physisorption was not significantly different from that of untreated titanium at any time point, whereas BSP application via covalent coupling caused reduced cell adhesion during the first few hours in culture. Cell migration was increased on titanium disks that were treated with higher concentrations of BSP solution, independent of the coating method. During the early phases of hOB proliferation, a suppressive effect of BSP was observed independent of its concentration, particularly when BSP was applied to the titanium surface via physisorption. Although alkaline phosphatase activity was reduced in the BSP-coated titanium groups after 4 days in culture, increased calcium deposition was observed after 21 days. In particular, the gene expression level of RUNX2 was upregulated by BSP. The increase in calcium deposition and the stimulation of cell differentiation induced by BSP highlight its potential as a surface modifier that could enhance the osseointegration of orthopedic implants. Both physisorption and covalent coupling of BSP are similarly effective, feasible methods, although a higher BSP concentration is recommended.

Type, density, and presentation of grafted adhesion peptides on polysaccharide-based hydrogels control preosteoblast behavior and differentiation.

In this work, cell-responsive polysaccharide hydrogels were prepared by a simple procedure based on the sequential bioconjugation and cross-linking of the polysaccharide backbone with bioactive peptides and poly(ethylene glycol)-bis(thiol) (PEG-(SH)2), respectively. Using thiol-ene reactions, we successfully functionalized hyaluronic acid (HA) and carboxymethylcellulose (CMC) with short and long peptides (5-mer and 15-mer derivatives, respectively) derived from adhesive proteins of bone extracellular matrix. The resulting HA-peptide and CMC-peptide conjugates with varying degrees of substitution were then carefully characterized by (1)H NMR spectroscopy to precisely control the peptide density into the hydrogels cross-linked with PEG-(SH)2. Preosteoblast seeded on the hydrogels with controlled identical stiffness spread in a manner that was strongly dependent on ligand density. Surprisingly, increasing the density of the adhesive peptide anchors did not result in a plateau of initial cell spreading but rather in a bell-shaped cell response that varies with the nature of both polysaccharide backbone and functional peptide. Placing the cells under optimal conditions for cell/hydrogel interaction, we showed that in HA hydrogels, the polysaccharide moiety is not solely a passive scaffold that presents the active peptides but is an active player in cell microenvironment to control and sustain cell activity.

Structure-activity relationship of human bone sialoprotein peptides.

In the current study, the relationship between the structure of the RGD-containing human bone sialoprotein (hBSP) peptide 278-293 and its attachment activity toward osteoblast-like (MC3T3) cells was investigated. This goal was accomplished by examining the comparative cell-attachment activities of several truncated forms of peptide 278-293. Computer modeling of the various peptides was also performed to assess the role of secondary structure in peptide bioactivity. Elimination of tyrosine-278 at the N-terminus resulted in a more dramatic loss of cell-attachment activity compared with the removal of either tyrosine-293 or the arg-ala-tyr (291-293) tripeptide. Although replacement of the RGD (arg-gly-asp) peptide moiety with peptide KAE (lys-ala-glu) resulted in a dramatic loss of cell-attachment activity, a peptide containing RGE (arg-gly-glu) in place of RGD retained 70-85% of the parental peptide's attachment activity. These results suggest that the N-terminal RGD-flanking region of hBSP peptide 278-293, in particular the tyrosine-278 residue, represents a second cell-attachment site that stabilizes the RGD-integrin receptor complex. Computer modeling also suggested that a ß-turn encompassing RGD or RGE in some of the hBSP peptides may facilitate its binding to integrins by increasing the exposure of the tripeptide. This knowledge may be useful in the future design of biomimetic peptides which are more effective in promoting the attachment of osteogenic cells to implant surfaces in vivo.

Biological effects of functionalizing copolymer scaffolds with nanodiamond particles.

Significant evidence has indicated that poly(L-lactide)-co-(ɛ-caprolactone) [(poly(LLA-co-CL)] scaffolds could be one of the suitable candidates for bone tissue engineering. Oxygen-terminated nanodiamond particles (n-DP) were combined with poly(LLA-co-CL) and revealed to be positive for cell growth. In this study, we evaluated the influence of poly(LLA-co-CL) scaffolds modified by n-DP on attachment, proliferation, differentiation of bone marrow stromal cells (BMSCs) in vitro, and on bone formation using a sheep calvarial defect model. BMSCs were seeded on either poly(LLA-co-CL)- or n-DP-coated scaffolds and incubated for 1 h. Scanning electron microscopy (SEM) and fluorescence microscopy were used in addition to protein and DNA measurements to evaluate cellular attachment on the scaffolds. To determine the effect of n-DP on proliferation of BMSCs, cell/scaffold constructs were harvested after 3 days and evaluated by Bicinchoninic Acid (BCA) protein assay and SEM. In addition, the osteogenic differentiation of cells grown for 2 weeks on the various scaffolds and in a dynamic culture condition was evaluated by real-time RT-PCR. Unmodified and modified scaffolds were implanted into the calvaria of six-year-old sheep. The expression of collagen type I (COL I) and bone morphogenetic protein-2 (BMP-2) after 4 weeks as well as the formation of new bone after 12 and 24 weeks were analyzed by immunohistochemistry and histology. Scaffolds modified with n-DP supported increased cell attachment and the mRNA expression of osteopontin (OPN), bone sialoprotein (BSP), and BMP-2 were significantly increased after 2 weeks of culture. The BMSCs had spread well on the various scaffolds investigated after 3 days in the study with no significant difference in cell proliferation. Furthermore, the in vivo data revealed more positive staining of COL I and BMP-2 in relation to the n-DP-coated scaffolds after 4 weeks and presented more bone formation after 12 and 24 weeks. n-DP modification significantly increased cell attachment and differentiation of BMSCs on poly(LLA-co-CL) scaffolds in vitro and enhanced bone formation in vivo.

In vivo biological responses to silk proteins functionalized with bone sialoprotein.

Recombinant 6mer + BSP protein, combining six repeats of the consensus sequence for Nephila clavipes dragline (6mer) and bone sialoprotein sequence (BSP), shows good support for cell viability and induces the nucleation of hydroxyapatite and tricalcium phosphate during osteoblast in vitro culture. The present study is conducted to characterize this bioengineered protein-based biomaterial further for in vivo behavior related to biocompatibility. 6mer + BSP protein films are implanted in subcutaneous pouches in the back of mice and responses are evaluated by flow cytometry and histology. The results show no major differences between the inflammatory responses induced by 6mer + BSP films and the responses observed for the controls. Thus, this new chimeric protein could represent an alternative for bone regeneration applications.

A structural and functional model for human bone sialoprotein.

Human bone sialoprotein (BSP) is an essential component of the extracellular matrix of bone. It is thought to be the primary nucleator of hydroxyapatite crystallization, and is known to bind to hydroxyapatite, collagen, and cells. Mature BSP shows extensive post-translational modifications, including attachment of glycans, sulfation, and phosphorylation, and is highly flexible with no specific 2D or 3D structure in solution or the solid state. These features have severely limited the experimental characterization of the structure of this protein. We have therefore developed a 3D structural model for BSP, based on the available literature data, using molecular modelling techniques. The complete model consists of 301 amino acids, including six phosphorylated serines and two sulfated tyrosines, plus 92 N- and O-linked glycan residues. A notable feature of the model is a large acidic patch that provides a surface for binding Ca(2+) ions. Density functional theory quantum calculations with an implicit solvent model indicate that Ca(2+) ions are bound most strongly by the phosphorylated serines within BSP, along with reasonably strong binding to Asp and Glu, but weak binding to His and sulfated tyrosine. The process of early hydroxyapatite nucleation has been studied by molecular dynamics on an acidic surface loop of the protein; the results suggest that the cationic nature of the loop promotes nucleation by attracting Ca(2+) ions, while its flexibility allows for their rapid self-assembly with PO(4)(3-) ions, rather than providing a regular template for crystallization. The binding of a hydroxyapatite crystal at the protein's acidic patch has also been modelled. The relationships between hydroxyapatite, collagen and BSP are discussed.

Enhanced osteogenesis by collagen-binding peptide from bone sialoprotein in vitro and in vivo.

Bone sialoprotein (BSP) is a mineralized, tissue-specific, and noncollagenous protein. The binding of BSP to collagen is thought to be important for the initiation of bone mineralization and formation. In this study, we elucidated the osteogenic efficiency of the collagen-binding (CB) peptide derived from BSP in vitro and in vivo. The CB peptide increased osteoblastic differentiation marker gene and protein expression without affecting cell proliferation. The osteoblastic differentiation by the CB peptide is performed by the activation of extracellular signal-regulated kinase (ERK1/2) and protein kinase B (Akt). Notably, the activation of CB peptide-induced osteogenic differentiation was completely blocked to the basal level by the specific inhibitors for ERK1/2 (U0126) and Akt (LY294002). In vivo results further demonstrated that the CB peptide-coated hydroxyapatite scaffold was able to induce bone formation in the bone defect. Taken together, the CB peptide can be developed as an osteoblastic differentiation agent as well as a fusion biomaterial for bone regeneration therapy.

Effect of BMP-2 and/or BMP-9 on preosteoblasts attached to polycaprolactone functionalized by adhesive peptides derived from bone sialoprotein.

Biomaterials functionalized by adhesive peptides improve the cell-substratum interaction. However, their influence on the response of cells to growth factors is still poorly understood. We have shown that bone morphogenetic protein (BMP) 2 activates the Smad pathway only in murine MC3T3-E1 preosteoblasts attached to polycaprolactone (PCL) film functionalized by RGD peptides derived from bone sialoprotein (pRGD). We have now analysed the way recombinant human BMP-2 and/or BMP-9 (0.38 nM) influence the signal transduction and differentiation of MC3T3-E1 preosteoblasts attached to PCL-pRGD. While kinetics of MAPK activation were similar in cells treated by BMP-2 and BMP-9, different kinetics of Smad activation and ß-catenin stabilization were observed. BMP-2 induced Smad1/5/8 phosphorylation within 0.5 and BMP-9 within 4 h, while the ß-catenin was lower at 2 h only in cells treated with BMP-9. However, both BMPs induced the translocation of phosphorylated Smad1/5/8 to the nucleus at 4 h and increased Dlx5, osterix and osteocalcin transcripts as well as alkaline phosphatase activity at 72 h. A BMP-2/BMP-9 combination that maintained the ß-catenin amount constant but reduced that of phosphorylated Smad within 4 h had quite similar effect than BMP-2 alone. It is therefore important to determine how biomimetic materials influence the response of cells to BMPs.

Determination of osteogenic or adipogenic lineages in muscle-derived stem cells (MDSCs) by a collagen-binding peptide (CBP) derived from bone sialoprotein (BSP).

Bone sialoprotein (BSP) is a mineralized, tissue-specific, non-collagenous protein that is normally expressed only in mineralized tissues such as bone, dentin, cementum, and calcified cartilage, and at sites of new mineral formation. The binding of BSP to collagen is thought to be important for initiating bone mineralization and bone cell adhesion to the mineralized matrix. Several recent studies have isolated stem cells from muscle tissue, but their functional properties are still unclear. In this study, we examined the effects of a synthetic collagen-binding peptide (CBP) on the differentiation efficiency of muscle-derived stem cells (MDSCs). The CBP sequence (NGVFKYRPRYYLYKHAYFYPHLKRFPVQ) corresponds to residues 35-62 of bone sialoprotein (BSP), which are located within the collagen-binding domain in BSP. Interestingly, this synthetic CBP inhibited adipogenic differentiation but increased osteogenic differentiation in MDSCs. The CBP also induced expression of osteoblastic marker proteins, including alkaline phosphatase (ALP), type I collagen, Runt-related transcription factor 2 (Runx2), and osteocalcin; prevented adipogenic differentiation in MDSCs; and down-regulated adipose-specific mRNAs, such as adipocyte protein 2 (aP2) and peroxisome proliferator-activated receptor γ. The CBP increased Extracellular signal-regulated kinases (ERK) 1/2 protein phosphorylation, which is important in lineage determination. These observations suggest that this CBP determines the osteogenic or adipogenic lineage in MDSCs by activating ERK1/2. Taken together, a novel CBP could be a useful candidate for regenerating bone and treating osteoporosis, which result from an imbalance in osteogenesis and adipogenesis differentiation.

Theoretical study of bone sialoprotein in bone biomineralization.

Bone sialoprotein (BSP) is an acidic, non-collagenous protein specific to bone proposed previously to promote hydroxyapatite (HAP) nucleation and modulate HAP nanocrystal growth. Specifically, two phosphorylated acidic amino acid sequences in BSP, highly conserved across several vertebrates, are the proposed active sites. We selected one of these sites, i.e. (Sp)(2)E(8), where Sp represents a phosphoserine as a model peptide to study the role of BSP. We used molecular dynamics simulations to determine whether an α-helix or a random coil peptide conformation promotes templated HAP nucleation. A bioinformatics method helps infer preferential crystal growth directions by predicting the likely peptide conformations adsorbed on the (001), (100), and (110) crystal faces of HAP. Results suggest that, independent of conformation, no stable nucleating template is formed and, thus, the ion distributions in the vicinity of the peptide that eventually lead to a stable nucleus start out with disordered arrangements of ions. When adsorbed on all three faces, the Sp residues bind strongly regardless of the peptide conformation, and the Glu residues show different propensities to form helical conformations. The lack of geometrical templating between the peptide residues and all HAP surface sites indicates that adsorption and subsequent crystal growth modulation may be structurally nonspecific.

TiO2 -enriched polymeric powder coatings support human mesenchymal cell spreading and osteogenic differentiation.

Novel polymeric powder coatings (PPC) were prepared by ultrafine powder coating technology and shown to support human mesenchymal cell attachment and growth. PPC surfaces enriched with nano-TiO(2) (nTiO(2)) showed enhanced cellular responses, and were compared to commercially pure titanium (cpTi). After cell attachment and growth, osteogenic differentiation and bone matrix formation ensures osseointegration for implantable biomaterials. Therefore, the objective of this study was to determine if mesenchymal cells grown on PPC could undergo osteogenic differentiation by inducing Runx2 and bone matrix proteins, and then initiate mineralization. Atomic force microscopy revealed intricate three-dimensional micro-topographies, and the measures of nano-roughness and porosity were similar for all PPC surfaces. Scanning electron microscopy showed that the cells attached and spread out over all of the surfaces. After 1 week in osteogenic media, RT-PCR analysis showed the induction of Runx2, the up-regulation of type I collagen, and the initial detection of alkaline phosphatase and bone sialoprotein. After 4 weeks, Alizarin Red staining showed mineral deposition. However, cell spreading and osteogenic differentiation were significantly (P < 0.05) higher on the cpTi controls than on the PPC surfaces. Furthermore, spreading and differentiation were consistently higher on the titanium-enriched PPC-2, -3 and -4 than on the titanium-free PPC-1. Therefore, despite the presence of complex micro-topographies and nano-features, titanium-enrichment enhanced the cellular response, and pure titanium still provided the best substrate. These findings confirm the cytocompatibility of these novel polymeric coatings and suggest that titanium-enrichment and nTiO(2) additives may enhance their performance.

Modification of polymer networks with bone sialoprotein promotes cell attachment and spreading.

Biomaterials used for tissue engineering scaffolds act as temporary substrates, on which cells deposit newly synthesized extracellular matrix. In cartilage tissue engineering, polycaprolactone/poly(2-hydroxyethyl methacrylate) (PCL/pHEMA) polymer blends have been used as scaffold materials, but their use in osseous tissue engineering has been more limited. The objective of this study was to evaluate modification of PCL/pHEMA surfaces with bone sialoprotein (BSP), an extracellular matrix protein important in regulating osseous tissue formation. Modification of surfaces with BSP significantly enhanced osteoblastic cell attachment and spreading, without compromising proliferation. Thus, BSP-immobilization may be a useful strategy for optimizing scaffolds for skeletal tissue engineering.