Microarray-based gene expression analysis of human osteoblasts in response to different biomaterials

Several biomaterials have been widely used in bone regeneration/substitution procedures in orthopedic and oral surgery. However, how these biomaterials alter osteoblast gene expression is poorly understood. We therefore attempted to address this question by using cDNA microarray technique to identify genes that are differentially regulated in osteoblasts exposed to biomaterials comprehending the biocompatibility spectrum of bioactive (bioglass and hydroxyapatite), bioinert (Ti and stainless steel), and biotolerant (polymethylmethacrylate). By using a cDNA microarray containing 687 human IMAGE sequences, we identified in primary cultures of osteoblastic cells differentiated from the human bone marrow and exposed to these biomaterials, genes whose expression was significantly upregulated or downregulated. Among the differentially expressed genes we have found those involved with cell cycle regulation, cell differentiation and proliferation, apoptosis, cell adhesion, bone mineralization and skeletal development. These results can be relevant to a better understanding of the molecular mechanism underlying the behavior of osteoblasts in bone regenerative procedures.     

Biological behavior of pre-osteoblasts on natural hydroxyapatite: a study of signaling molecules from attachment to differentiation

Several biomaterials have been widely used in bone regeneration in both orthopedic and oral surgeries. However, it is poorly understood how these biomaterials alter osteoblast phenotype. It prompted us to examine the involvement of signaling proteins during preosteoblast adhesion (attachment), proliferation, and differentiation on natural hydroxyapatite (HA) from bovine bone. Our results indicated that natural HA is able to promote osteoblast adhesion, proliferation, and differentiation. The osteoblast/HA interaction requires phosphorylation of tyrosine residues of focal adhesion kinase, Src, and Paxillin upon integrin activation, which culminates in the control of cofilin phosphorylation (at serine 03) via rac-1 activation. In part, these signaling pathways are responsible for actin-rearrangement, responsible to adapt cell-shape on HA particles. In regarding to osteoblast differentiation, we showed that natural HA favored extracellular matrix remodeling by stimulating matrix metalloproteinase activities and alkaline phosphatase activity. Overall, this study demonstrates that osteoblast response toward bovine bone HA is initially mediated by activation of focal adhesion components, culminating on actin-rearrangement executed by cofilin activation via rac-1. Moreover, bovine bone HA provided an excellent microenvironment for osteoblast activity, since adhesion to differentiation.     

Current views on calcium phosphate osteogenicity and the translation into effective bone regeneration strategies

Calcium phosphate (CaP) has traditionally been used for the repair of bone defects because of its strong resemblance to the inorganic phase of bone matrix. Nowadays, a variety of natural or synthetic CaP-based biomaterials are produced and have been extensively used for dental and orthopaedic applications. This is justified by their biocompatibility, osteoconductivity and osteoinductivity (i.e. the intrinsic material property that initiates de novo bone formation), which are attributed to the chemical composition, surface topography, macro/microporosity and the dissolution kinetics. However, the exact molecular mechanism of action is unknown. This review paper first summarizes the most important aspects of bone biology in relation to CaP and the mechanisms of bone matrix mineralization. This is followed by the research findings on the effects of calcium (Ca²⁺) and phosphate (${\text{PO}}_4^{3-}$) ions on the migration, proliferation and differentiation of osteoblasts during in vivo bone formation and in vitro culture conditions. Further, the rationale of using CaP for bone regeneration is explained, focusing thereby specifically on the material’s osteoinductive properties. Examples of different material forms and production techniques are given, with the emphasis on the state-of-the art in fine-tuning the physicochemical properties of CaP-based biomaterials for improved bone induction and the use of CaP as a delivery system for bone morphogenetic proteins. The use of computational models to simulate the CaP-driven osteogenesis is introduced as part of a bone tissue engineering strategy in order to facilitate the understanding of cell–material interactions and to gain further insight into the design and optimization of CaP-based bone reparative units. Finally, limitations and possible solutions related to current experimental and computational techniques are discussed.     

Mediation of biomaterial-cell interactions by adsorbed proteins: a review

An appropriate cellular response to implanted surfaces is essential for tissue regeneration and integration. It is well described that implanted materials are immediately coated with proteins from blood and interstitial fluids, and it is through this adsorbed layer that cells sense foreign surfaces. Hence, it is the adsorbed proteins, rather than the surface itself, to which cells initially respond. Diverse studies using a range of materials have demonstrated the pivotal role of extracellular adhesion proteins--fibronectin and vitronectin in particular--in cell adhesion, morphology, and migration. These events underlie the subsequent responses required for tissue repair, with the nature of cell surface interactions contributing to survival, growth, and differentiation. The pattern in which adhesion proteins and other bioactive molecules adsorb thus elicits cellular reactions specific to the underlying physicochemical properties of the material. Accordingly, in vitro studies generally demonstrate favorable cell responses to charged, hydrophilic surfaces, corresponding to superior adsorption and bioactivity of adhesion proteins. This review illustrates the mediation of cell responses to biomaterials by adsorbed proteins, in the context of osteoblasts and selected materials used in orthopedic implants and bone tissue engineering. It is recognized, however, that the periimplant environment in vivo will differ substantially from the cell-biomaterial interface in vitro. Hence, one of the key issues yet to be resolved is that of the interface composition actually encountered by osteoblasts within the sequence of inflammation and bone regeneration.     

In vitro differentiation of human mesenchymal stem cells on Ti6Al4V surfaces

Long-term stability of arthroplasty prosthesis depends on the integration between the bone tissue and the implanted biomaterials, which requires the contribution of osteoblastic precursors and their continuous differentiation into the osteoblastic phenotype. Classically, these interactions are tested in vitro using mesenchymal stem cells (MSCs) isolated and ex vivo expanded from bone marrow aspirates. Human adipose tissue-derived stromal cells (AMSCs) may be a more convenient source of MSCs, according to their abundance and accessibility, but no data are available on their in vitro interactions with hard biomaterials. The aim of this work is to compare the osteogenic potential of human AMSCs and bone marrow-derived MSCs (BMMSCs) and to evaluate their response to Ti6Al4V alloy in terms of adhesion, proliferation and differentiation features, using the human osteosarcoma cell line SaOS-2 for comparison. The overall results showed that AMSCs have the same ability to produce bone matrix as BMMSCs and that Ti6Al4V surfaces exhibit an osteoinductive action on AMSCs, promoting their differentiation into functional osteoblasts and increasing bone formation. In conclusion, adipose tissue is a promising autologous source of osteoblastic cells with important clinical implications for bone tissue engineering.     

Gradient biomaterials for soft-to-hard interface tissue engineering

Interface tissue engineering (ITE) is a rapidly developing field that aims to fabricate biological tissue alternates with the goal of repairing or regenerating the functions of diseased or damaged zones at the interface of different tissue types (also called "interface tissues"). Notable examples of the interface tissues in the human body include ligament-to-bone, tendon-to-bone and cartilage-to-bone. Engineering interface tissues is a complex process, which requires a combination of specialized biomaterials with spatially organized material composition, cell types and signaling molecules. Therefore, the use of conventional biomaterials (monophasic or composites) for ITE has certain limitations to help stimulate the tissue integration or recreating the structural organization at the junction of different tissue types. The advancement of micro- and nanotechnologies enable us to develop systems with gradients in biomaterials properties that encourage the differentiation of multiple cell phenotypes and subsequent tissue development. In this review we discuss recent developments in the fabrication of gradient biomaterials for controlling cellular behavior such as migration, differentiation and heterotypic interactions. Moreover, we give an overview of potential uses of gradient biomaterials in engineering interface tissues such as soft tissues (e.g. cartilage) to hard tissues (e.g. bone), with illustrated experimental examples. We also address fundamentals of interface tissue organization, various gradient biomaterials used in ITE, micro- and nanotechnologies employed for the fabrication of those gradients, and certain challenges that must be met in order for ITE to reach its full potential.     

Behaviour of mesenchymal stem cells, fibroblasts and osteoblasts on smooth surfaces

Understanding of the interactions between cells and surfaces is essential in the field of tissue engineering and biomaterials. This study aimed to compare the adhesion, proliferation and differentiation of human mesenchymal stem cells (hMSCs), an osteoblast cell line (MC3T3-E1) and gingival fibroblasts (HGF-1) on tissue culture polystyrene (TCPS), glass and titanium (Ti). The average surface roughness was 5, 0.2 and 40×10(-3) μm for TCPS, glass and Ti, respectively. Immunocytochemistry and image analysis made it possible to quantify the number and morphology of adherent cells as well as the density of the focal points. Regardless of the substrate, both hMSCs and osteoblastic cells were mainly branch-shaped. HGF-1 exhibited a significantly higher number of focal points on Ti than on TCPS and glass. Alizarin red quantification indicated that both hMSCs and osteoblastic cells were more differentiated on TCPS than on Ti and glass. The surface properties of substrates, such as roughness, wettability and chemical composition, modulated the behaviour of the cells. Early events, such as cell adhesion, may influence the differentiation of hMSC and consequently tissue healing around implanted biomaterials.     

Biomimetic approaches to modulate cellular adhesion in biomaterials: A review

Natural extracellular matrix (ECM) proteins possess critical biological characteristics that provide a platform for cellular adhesion and activation of highly regulated signaling pathways. However, ECM-based biomaterials can have several limitations, including poor mechanical properties and risk of immunogenicity. Synthetic biomaterials alleviate the risks associated with natural biomaterials but often lack the robust biological activity necessary to direct cell function beyond initial adhesion. A thorough understanding of receptor-mediated cellular adhesion to the ECM and subsequent signaling activation has facilitated development of techniques that functionalize inert biomaterials to provide a biologically active surface. Here we review a range of approaches used to modify biomaterial surfaces for optimal receptor-mediated cell interactions, as well as provide insights into specific mechanisms of downstream signaling activation. In addition to a brief overview of integrin receptor-mediated cell function, so-called “biomimetic” techniques reviewed here include (i) surface modification of biomaterials with bioadhesive ECM macromolecules or specific binding motifs, (ii) nanoscale patterning of the materials and (iii) the use of “natural-like” biomaterials.     

Osteoblastic cell response to thin film of poorly crystalline calcium phosphate apatite formed at low temperatures

The response of osteoblastic cells to a thin film of poorly crystalline calcium phosphate apatite crystals (PCA) was examined in vitro. The PCA thin film was prepared on polystyrene culture dishes using highly metastable calcium phosphate ion solution at low temperatures. The PCA thin film was formed through fusion and transformation of granular calcium phosphate particles, which had initially formed on the surface, into a film of calcium phosphate apatite crystal. The PCA thin film was used for cell culture without additional surface treatment. The osteoblastic cell behaviors including adhesion, proliferation, expression of the marker genes, and calcified matrix formation were examined on the PCA thin film using primary osteoblasts or MC3T3-E1 cells. The cells were well attached and had spread in a slender shape over the PCA thin film. The extent of cell proliferation on the PCA thin film is as much as on the plain dishes. In addition, a much larger number of calcified nodules had formed on the PCA thin film than on the plain dish. The expression of the marker genes such as alkaline phosphatase, osteocalcin, osteopontin, osteonectin was apparent. These results demonstrate that the osteoblasts exhibit a full spectrum of cellular activity including the adequate differentiation on the PCA thin film. Therefore, a PCA thin film can be used as a coating material for biomaterials where the surface is not adequate for inducing the full activity of bone cells.     

Fabrication and characterization of chitosan/OGP coated porous poly(ε-caprolactone) scaffold for bone tissue engineering

As one of the stimulators on bone formation, osteogenic growth peptide (OGP) improves both proliferation and differentiation of the bone cells in vitro and in vivo. The aim of this work was the preparation of three dimensional porous poly(ε-caprolactone) (PCL) scaffold with high porosity, well interpore connectivity, and then its surface was modified by using chitosan (CS)/OGP coating for application in bone regeneration. In present study, the properties of porous PCL and CS/OGP coated PCL scaffold, including the microstructure, water absorption, porosity, hydrophilicity, mechanical properties, and biocompatibility in vitro were investigated. Results showed that the PCL and CS/OGP-PCL scaffold with an interconnected network structure have a porosity of more than 91.5, 80.8%, respectively. The CS/OGP-PCL scaffold exhibited better hydrophilicity and mechanical properties than that of uncoated PCL scaffold. Moreover, the results of cell culture test showed that CS/OGP coating could stimulate the proliferation and growth of osteoblast cells on CS/OGP-PCL scaffold. These finding suggested that the surface modification could be a effective method on enhancing cell adhesion to synthetic polymer-based scaffolds in tissue engineering application and the developed porous CS/OGP-PCL scaffold should be considered as alternative biomaterials for bone regeneration.     

Bioactive membranes for bone regeneration applications: Effect of physical and biomolecular signals on mesenchymal stem cell behavior

This study focuses on the in vitro characterization of bioactive elastin-like recombinamer (ELR) membranes for bone regeneration applications. Four bioactive ELRs exhibiting epitopes designed to promote mesenchymal stem cell adhesion (RGDS), endothelial cell adhesion (REDV), mineralization (HAP), and both cell adhesion and mineralization (HAP-RGDS) were synthesized using standard recombinant protein techniques. The materials were then used to fabricate ELR membranes incorporating a variety of topographical micropatterns including channels, holes and posts. Primary rat mesenchymal stem cells (rMSCs) were cultured on the different membranes and the effects of biomolecular and physical signals on cell adhesion, morphology, proliferation, and differentiation were evaluated. All results were analyzed using a custom-made MATLAB program for high throughput image analysis. Effects on cell morphology were mostly dependent on surface topography, while cell proliferation and cell differentiation were largely dependent on the biomolecular signaling from the ELR membranes. In particular, osteogenic differentiation (evaluated by staining for the osteoblastic marker osterix) was significantly enhanced on cells cultured on HAP membranes. Remarkably, cells growing on membranes containing the HAP sequence in non-osteogenic differentiation media exhibited significant up-regulation of the osteogenic marker as early as day 5, while those growing on fibronectin-coated glass in osteogenic differentiation media did not. These results are part of our ongoing effort to develop an optimized molecularly designed periosteal graft.     

Octacalcium phosphate: Osteoconductivity and crystal chemistry

Octacalcium phosphate (OCP), which is structurally similar to hydroxyapatite (HA), is a possible precursor of bone apatite crystals. Although disagreement remains as to whether OCP comprises the initial mineral crystals in the early stage of bone mineralization, the results of recent biomaterial studies using synthetic OCP indicate the potential role of OCP as a bone substitute material, owing to its highly osteoconductive and biodegradable characteristics. OCP tends to convert to HA not only in an in vitro environment, but also as an implant in bone defects. Several lines of evidence from both in vivo and in vitro studies suggest that the conversion process could be involved in the stimulatory capacity of OCP for osteoblastic differentiation and osteoclast formation. However, the osteoconductivity of OCP cannot always be secured if an OCP with distinct crystal characteristics is used, because the stoichiometry and microstructure of OCP crystals greatly affect bone-regenerative properties. Osteoconductivity and stimulatory capabilities may be caused by the chemical characteristics of OCP, which allows the release or exchange of calcium and phosphate ions with the surrounding of this salt, and its tendency to grow towards specific crystal faces, which could be a variable of the synthesis condition. This paper reviews the effect of calcium phosphates on osteoblastic activity and bone regeneration, with a special emphasis on OCP, since OCP seems to be performing better than other calcium phosphates in vivo.     

Nonwoven membranes for tissue engineering: an overview of cartilage,epithelium, and bone regeneration

Scaffold-type biomaterials are crucial for application in tissue engineering. Among them, the use of a nonwoven scaffold has grown in recent years and has been widely investigated for the regeneration of different types of tissues. Several polymers, whether they are synthetic, biopolymers or both, have been used to produce a scaffold that can mimic the natural tissue to which it will be applied to. The scaffolds used in tissue engineering must be biocompatible and allow cell adhesion and proliferation to be applied in tissue engineering. In addition, the scaffolds should maintain the mechanical properties and architecture of the desired tissue. Nonwoven fabrics have produced good results and are more extensively applied for the regeneration of cartilage, epithelial and bone tissues. Recent advances in tissue engineering have shown promising results, however, no ideal material or standardization parameters and characteristics of the materials were obtained. The present review provides an overview of the application of nonwoven scaffolds, including the main results obtained regarding the properties of the biomaterials and their applications in vitro and in vivo, focusing on the cartilaginous, the epithelium, and bone tissue regeneration.     

Surface modifications by gas plasma control osteogenic differentiation of MC3T3-E1 cells

Numerous studies have shown that the physicochemical properties of biomaterials can control cell activity. Cell adhesion, proliferation, differentiation as well as tissue formation in vivo can be tuned by properties such as the porosity, surface micro- and nanoscale topography and chemical composition of biomaterials. This concept is very appealing for tissue engineering since instructive properties in bioactive materials can be more economical and time efficient than traditional strategies of cell pre-differentiation in vitro prior to implantation. The biomaterial surface, which is easy to modify due to its accessibility, may provide the necessary signals to elicit a certain cellular behavior. Here, we used gas plasma technology at atmospheric pressure to modify the physicochemical properties of polylactic acid and analyzed how this influenced pre-osteoblast proliferation and differentiation. Tetramethylsilane and 3-aminopropyl-trimethoxysilane with helium as a carrier gas or a mixture of nitrogen and hydrogen were discharged to polylactic acid discs to create different surface chemical compositions, hydrophobicity and microscale topographies. Such modifications influenced protein adsorption and pre-osteoblast cell adhesion, proliferation and osteogenic differentiation. Furthermore polylactic acid treated with tetramethylsilane enhanced osteogenic differentiation compared to the other surfaces. This promising surface modification could be further explored for potential development of bone graft substitutes.     

A theranostic agent to enhance osteogenic and magnetic resonance imaging properties of calcium phosphate cements

With biomimetic biomaterials, like calcium phosphate cements (CPCs), non-invasive assessment of tissue regeneration is challenging. This study describes a theranostic agent (TA) to simultaneously enhance both imaging and osteogenic properties of such a bone substitute material. For this purpose, mesoporous silica beads were produced containing an iron oxide core to enhance bone magnetic resonance (MR) contrast. The same beads were functionalized with silane linkers to immobilize the osteoinductive protein BMP-2, and finally received a calcium phosphate coating, before being embedded in the CPC. Both in vitro and in vivo tests were performed. In vitro testing showed that the TA beads did not interfere with essential material properties like cement setting. Furthermore, bioactive BMP-2 could be efficiently released from the carrier-beads. In vivo testing in a femoral condyle defect rat model showed long-term MR contrast enhancement, as well as improved osteogenic capacity. Moreover, the TA was released during CPC degradation and was not incorporated into the newly formed bone. In conclusion, the described TA was shown to be suitable for longitudinal material degradation and bone healing studies.     

MG63 osteoblastic cell adhesion to the hydrophobic surface precoated with recombinant osteopontin fragments

The hydrophobicity of biomaterials has been recognized as a limitation to the adequate function of anchorage-dependent cells when hydrophobic biomaterials are used for tissue engineering. This is due to flawed solid-state signals from cell adhesion. In this study, a recombinant osteopontin (rOPN17-169) fragment containing the cell adhesion motifs was expressed in E. coli and was precoated on the hydrophobic surface prior to osteoblastic MG63 cell culture. Precoating the hydrophobic surface with rOPN17-169 improved osteoblastic cell adhesion, which was blocked by soluble RGDS. The adhesion of MG63 cells to rOPN17-169 pre-coated surface-activated mitogen-activated protein kinases (MAPK) such as extracellular signal-receptor kinase 1/2, p38, and c-Jun N-terminal kinase (JNK). In addition, p38 MAPK was activated in response to a soluble factor of transforming growth factor-beta in the cells adhered to the hydrophobic surface via rOPN17-169. This suggests that rOPN17-169 precoated on the hydrophobic surface can allow osteoblastic cells to generate adhesion signals sufficient for cell adhesion, MAPK activation, and the cytokine activation of osteoblastic cells.     

Regenerative potential of glycosaminoglycans for skin and bone

To meet the growing need for tissue replacement materials for our aging population, the development of new adaptive biomaterials is essential. The tissues with the highest demand for implant materials are skin and bone. These tissues share various similarities, including signaling pathways and extracellular matrix composition. Glycosaminoglycans such as hyaluronan and chondroitin sulfate are the major organic extracellular matrix components. They modulate the attraction of skin and bone precursor cells and their subsequent differentiation and gene expression and regulate the action of proteins essential to bone and skin regeneration. The precise action of glycosaminoglycans varies according to their structural composition mainly in respect to the degree of sulfation and polymer length. Changes in the glycosaminoglycan composition are frequently seen in physiological and pathological remodeling processes, such as bone formation or scaring. Here, we review the current state of knowledge of how the most common glycosaminoglycan, chondroitin sulfate and hyaluronan, interact with bone and skin cells, and summarize their potential in tissue engineering for skeletal and skin diseases.     

Synthetic peptide-coated bone mineral for enhanced osteoblastic activation in vitro and in vivo

A 15-mer synthetic peptide, designated P1, was derived from the bone morphogenetic protein (BMP) receptor I and BMP receptor II binding domains of BMP-2 for the purpose of enhancing bone regeneration capacity of inorganic bovine bone mineral. A second peptide, denoted P2, was designed by adding seven glutamic acid residues to the N-terminal of P1 to increase the surface coating efficiency onto bone mineral. The coating efficiency of P1 increased with the concentration of peptide. P2 peptide, in contrast, had a higher coating efficiency at lower peptide concentrations. The peptides properly transduced intracellular signals properly via the Smad and ERK pathways, thereby increasing mineralization in vitro, implying that the peptides alone can induce osteoblastic differentiation. Adhesion of cells to bone mineral was greater when peptides were present than in bone mineral alone. P1- and P2-coated bone mineral increased osteoblastic differentiation, as demonstrated by ALPase activity. P1-coated bone mineral stimulated more new bone regeneration in bone defect sites after 2 weeks than the peptide-free control. These peptides, in combination with bone grafts or implants, have the potential to enhance osteoblastic differentiation and bone regeneration.     

Osteoblast adhesion on biomaterials

The development of tissue engineering in the field of orthopaedic surgery is now booming. Two fields of research in particular are emerging: the association of osteo-inductive factors with implantable materials; and the association of osteogenic stem cells with these materials (hybrid materials). In both cases, an understanding of the phenomena of cell adhesion and, in particular, understanding of the proteins involved in osteoblast adhesion on contact with the materials is of crucial importance. The proteins involved in osteoblast adhesion are described in this review (extracellular matrix proteins, cytoskeletal proteins, integrins, cadherins, etc.). During osteoblast/material interactions, their expression is modified according to the surface characteristics of materials. Their involvement in osteoblastic response to mechanical stimulation highlights the significance of taking them into consideration during development of future biomaterials. Finally, an understanding of the proteins involved in osteoblast adhesion opens up new possibilities for the grafting of these proteins (or synthesized peptide) onto vector materials, to increase their in vivo bioactivity or to promote cell integration within the vector material during the development of hybrid materials.