Advanced Glycation End Products Effects on Adipocyte Niche Stiffness and Cell Signaling.

Adipose tissue metabolism under hyperglycemia results in Type II diabetes (T2D). To better understand how the adipocytes function, we used a cell culture that was exposed to glycation by adding intermediate carbonyl products, which caused chemical cross-linking and led to the formation of advanced glycation end products (AGEs). The AGEs increased the cells and their niche stiffness and altered the rheological viscoelastic properties of the cultured cells leading to altered cell signaling. The AGEs formed concomitant with changes in protein structure, quantified by spectroscopy using the 8-ANS and Nile red probes. The AGE effects on adipocyte differentiation were viewed by imaging and evidenced in a reduction in cellular motility and membrane dynamics. Importantly, the alteration led to reduced adipogenesis, that is also measured by qPCR for expression of adipogenic genes and cell signaling. The evidence of alteration in the plasma membrane (PM) dynamics (measured by CTxB binding and NP endocytosis), also led to the impairment of signal transduction and a decrease in AKT phosphorylation, which hindered downstream insulin signaling. The study, therefore, presents a new interpretation of how AGEs affect the cell niche, PM stiffness, and cell signaling leading to an impairment of insulin signaling.

Nutrition Alters the Stiffness of Adipose Tissue and Cell Signaling.

Adipose tissue is a complex organ composed of various cell types and an extracellular matrix (ECM). The visceral adipose tissue (VAT) is dynamically altered in response to nutritional regimens that lead to local cues affecting the cells and ECM. The adipocytes are in conjunction with the surrounding ECM that maintains the tissue's niche, provides a scaffold for cells and modulates their signaling. In this study, we provide a better understanding of the crosstalk between nutritional regimens and the ECM's stiffness. Histological analyses showed that the adipocytes in mice fed a high-fat diet (HFD) were increased in size, while the ECM was also altered with changes in mass and composition. HFD-fed mice exhibited a decrease in elastin and an increase in collagenous proteins. Rheometer measurements revealed a stiffer ECM in whole tissue (nECM) and decellularized (deECM) in HFD-fed animals. These alterations in the ECM regulate cellular activity and influence their metabolic function. HFD-fed mice expressed high levels of the receptor for advanced-glycation-end-products (RAGE), indicating that AGEs might play a role in these processes. The cells also exhibited an increase in phosphoserine332 of IRS-1, a decrease in the GLUT4 transporter levels at the cells' membrane, and a consequent reduction in insulin sensitivity. These results show how alterations in the stiffness of ECM proteins can affect the mechanical cues transferred to adipocytes and, thereby, influence the adipocytes' functionality, leading to metabolic disorders.

How to follow lipid droplets dynamics during adipocyte metabolism.

Lipid droplets (LDs) are important cellular organelles due to their ability to accumulate and store lipids. LD dynamics are associated with various cellular and metabolic processes. Accurate monitoring of LD's size and shape is of prime importance as it indicates the metabolic status of the cells. Unintrusive continuous quantification techniques have a clear advantage in analyzing LDs as they measure and monitor the cells' metabolic function and droplets over time. Here, we present a novel machine-learning-based method for LDs analysis by segmentation of phase-contrast images of differentiated adipocytes (in vitro) and adipose tissue (in vivo). We developed a new workflow based on the ImageJ waikato environment for knowledge analysis segmentation plugin, which provides an accurate, label-free, live single-cell, and organelle quantification of LD-related parameters. By applying the new method on differentiating 3T3-L1 cells, the size of LDs was analyzed over time in differentiated adipocytes and their correlation with other morphological parameters. Moreover, we analyzed the LDs dynamics during catabolic changes such as lipolysis and lipophagy and demonstrated its ability to identify different cellular subpopulations based on their structural, numerical, and spatial variability. This analysis was also implemented on unstained ex vivo adipose tissues to measure adipocyte size, an important readout of the tissue's metabolism. The presented approach can be applied in different LD-related metabolic conditions to provide a better understanding of LD biogenesis and function in vivo and in vitro while serving as a new platform that enables rapid and accurate screening of data sets.

Mesenchymal stem cell differentiation and usage for biotechnology applications: tissue engineering and food manufacturing.

Recent advances in the field of stem cell research now enable their utilisation for biotechnology applications in regenerative medicine and food tech. The first use of stem cells as biomedical devices employed a combination of cells and scaffold to restore, improve, or replace damaged tissues and to grow new viable tissue for replacement organs. This approach has also been adopted to replace meat production in the food industry. Mesenchymal stem cells are the source material used to induce cells to differentiate into the desired lineage. These technologies require mass propagation and rely on supplying the regulatory factors that direct differentiation. Mesenchymal stem cells can differentiate into fibroblastic and skeletal cells; fibroblastic/chondrogenic/osteogenic/myogenic and adipogenic lineages. Each differentiation fate requires specific key molecular regulators and appropriate activation conditions. Stem cell commitment determination involves a concerted effort of coordinated activation and silencing of lineage-specific genes. Transcription factors which bind gene promoters and chromatin-remodelling proteins are key players in the control process of lineage commitment and differentiation from embryogenesis through adulthood. Consequently, a major research challenge is to characterise such molecular pathways that coordinate lineage-specific differentiation and function. Revealing the mechanisms of action and the main factors will provide the knowledge necessary to control activation and regulation to achieve a specific lineage. Growing cells on a scaffold is a support system that mimics natural tissue and transduces the appropriate signals of the tissue niche for appropriate cellular function. The outcome of such research will deepen the understanding of cell differentiation to promote and advance the biotech, allowing the cell expansion required for their usage in therapy or the development of food tech.

Identification of UDP-Glucuronosyltransferase 2B15 (UGT2B15) as a Target for IGF1 and Insulin Action.

Normal growth and development in mammals are tightly controlled by numerous genetic factors and metabolic conditions. The growth hormone (GH)-insulin-like growth factor-1 (IGF1) hormonal axis is a key player in the regulation of these processes. Dysregulation of the GH-IGF1 endocrine system is linked to a number of pathologies, ranging from growth deficits to cancer. Laron syndrome (LS) is a type of dwarfism that results from mutation of the GH receptor (GHR) gene, leading to GH resistance and short stature as well as a number of metabolic abnormalities. Of major clinical relevance, epidemiological studies have shown that LS patients do not develop cancer. While the mechanisms associated with cancer protection in LS have not yet been elucidated, genomic analyses have identified a series of metabolic genes that are over-represented in LS patients. We hypothesized that these genes might constitute novel targets for IGF1 action. With a fold-change of 11.09, UDP-glucuronosyltransferase 2B15 (UGT2B15) was the top up-regulated gene in LS. The UGT2B15 gene codes for an enzyme that converts xenobiotic substances into lipophilic compounds and thereby facilitates their clearance from the body. We investigated the regulation of UGT2B15 gene expression by IGF1 and insulin. Both hormones inhibited UGT2B15 mRNA levels in endometrial and breast cancer cell lines. Regulation of UGT2B15 protein levels by IGF1/insulin, however, was more complex and not always correlated with mRNA levels. Furthermore, UGT2B15 expression was dependent on p53 status. Thus, UGT2B15 mRNA levels were higher in cell lines expressing a wild-type p53 compared to cells containing a mutated p53. Animal studies confirmed an inverse correlation between UGT2B15 and p53 levels. In summary, increased UGT2B15 levels in LS might confer upon patient's protection from genotoxic damage.

MYH10 Governs Adipocyte Function and Adipogenesis through Its Interaction with GLUT4.

Adipogenesis is dependent on cytoskeletal remodeling that determines and maintains cellular shape and function. Cytoskeletal proteins contribute to the filament-based network responsible for controlling the shape of adipocytes and promoting the intracellular trafficking of cellular components. Currently, the understanding of these mechanisms and their effect on differentiation and adipocyte function remains incomplete. In this study, we identified the non-muscle myosin 10 (MYH10) as a novel regulator of adipogenesis and adipocyte function through its interaction with the insulin-dependent glucose transporter 4 (GLUT4). MYH10 depletion in preadipocytes resulted in impaired adipogenesis, with knockdown cells exhibiting an absence of morphological alteration and molecular signals. MYH10 was shown in a complex with GLUT4 in adipocytes, an interaction regulated by insulin induction. The missing adipogenic capacity of MYH10 knockdown cells was restored when the cells took up GLUT4 vesicles from neighbor wildtype cells in a co-culture system. This signaling cascade is regulated by the protein kinase C ζ (PKCζ), which interacts with MYH10 to modify the localization and interaction of both GLUT4 and MYH10 in adipocytes. Overall, our study establishes MYH10 as an essential regulator of GLUT4 translocation, affecting both adipogenesis and adipocyte function, highlighting its importance in future cytoskeleton-based studies in adipocytes.

Exploring the Cell Stemness and the Complexity of the Adipose Tissue Niche.

Adipose tissue is a complex organ composed of different cellular populations, including mesenchymal stem and progenitor cells, adipocytes, and immune cells such as macrophages and lymphocytes. These cellular populations alter dynamically during aging or as a response to pathophysiology such as obesity. Changes in the various inflammatory cells are associated with metabolic complications and the development of insulin resistance, indicating that immune cells crosstalk with the adipocytes. Therefore, a study of the cell populations in the adipose tissue and the extracellular matrix maintaining the tissue niche is important for the knowledge on the regulatory state of the organ. We used a combination of methods to study various parameters to identify the composition of the resident cells in the adipose tissue and evaluate their profile. We analyzed the tissue structure and cells based on histology, immune fluorescence staining, and flow cytometry of cells present in the tissue in vivo and these markers' expression in vitro. Any shift in cells' composition influences self-renewal of the mesenchymal progenitors, and other cells affect the functionality of adipogenesis.

A Unique Marine-Derived Collagen: Its Characterization towards Biocompatibility Applications for Tissue Regeneration.

Biomedical engineering combines engineering and materials methods to restore, maintain, improve, or replace different types of biological tissues. In tissue engineering, following major injury, a scaffold is designed to support the local growth of cells, enabling the development of new viable tissue. To provide the conditions for the mechanical and structural properties needed for the restored tissue and its appropriate functioning, the scaffold requires specific biochemical properties in order to ensure a correct healing process. The scaffold creates a support system and requires a suitable material that will transduce the appropriate signals for the regenerative process to take place. A scaffold composed of material that mimics natural tissue, rather than a synthetic material, will achieve better results. Here, we provide an overview of natural components of marine-derived origin, the collagen fibers characterization schematic is summarized in the graphical abstract. The use of collagen fibers for biomedical applications and their performances in cell support are demonstrated in an in vitro system and in tissue regeneration in vivo.

Revealing Advanced Glycation End Products Associated Structural Changes in Serum Albumin.

Structural alterations in proteins have a significant impact on their function and body physiology. Glycation via nonenzymatic forms of cross-linking leads to proteins' conformational changes, the macromolecule being recognized as a stable fibrillary structure, oligomerization, and becoming advanced glycation end products (AGEs). Protein that undergoes glycation-related modifications, namely, ß-sheet enriched structural changes, are recognized as amyloid. In the current study, we characterized a single protein modified in vitro under physiological conditions to represent a protein glycation model. The glycation altered the helical conformation of serum albumin (SA) and promoted the formation of a ß-sheet enriched with amyloid fibrils detected at multidimensional levels. The nanoscale resolution by spectroscopy in the presence of thioflavin-T (ThT) and 8-anilinonaphthalene-1-sulfonic acid (8-ANS) showed binding of the fibrils formed in the presence of glucose (GLU) and the carbonyl metabolites methylglyoxal (MGO) and glycolaldehyde (GAD). In the presence of MGO and GAD, the SA becomes insoluble aggregates, demonstrated by TEM microscopy and dynamic light scattering (DLS). The protein oligomerization was visualized when separated via SDS gel electrophoresis and mass photometry (MP) assays. Following the glycation, eventually, the material polymerized and became stiffer. The level of stiffness was analyzed by a rheometer that revealed a quick alteration under MGO and GAD. This is the first study to combine multiple spectroscopy assays, imaging, and rheology measurements of SA and to demonstrate a resolution on a nanoscale structural toward better resolution of the conformational changes of glycated SA, oligomerization, and protein aggregations under physiological conditions.

Biocompatibility of a Marine Collagen-Based Scaffold In Vitro and In Vivo.

Scaffold material is essential in providing mechanical support to tissue, allowing stem cells to improve their function in the healing and repair of trauma sites and tissue regeneration. The scaffold aids cell organization in the damaged tissue. It serves and allows bio mimicking the mechanical and biological properties of the target tissue and facilitates cell proliferation and differentiation at the regeneration site. In this study, the developed and assayed bio-composite made of unique collagen fibers and alginate hydrogel supports the function of cells around the implanted material. We used an in vivo rat model to study the scaffold effects when transplanted subcutaneously and as an augment for tendon repair. Animals' well-being was measured by their weight and daily activity post scaffold transplantation during their recovery. At the end of the experiment, the bio-composite was histologically examined, and the surrounding tissues around the implant were evaluated for inflammation reaction and scarring tissue. In the histology, the formation of granulation tissue and fibroblasts that were part of the inclusion process of the implanted material were noted. At the transplanted sites, inflammatory cells, such as plasma cells, macrophages, and giant cells, were also observed as expected at this time point post transplantation. This study demonstrated not only the collagen-alginate device biocompatibility, with no cytotoxic effects on the analyzed rats, but also that the 3D structure enables cell migration and new blood vessel formation needed for tissue repair. Overall, the results of the current study proved for the first time that the implantable scaffold for long-term confirms the well-being of these rats and is correspondence to biocompatibility ISO standards and can be further developed for medical devices application.

Biomechanical stimulation effects on the metabolism of adipocyte.

Adipose tissue plays a leading role in obesity, which, in turn, can lead to Type 2 diabetes. Adipocytes (AD) respond to the biomechanical stimulation experienced in fat tissue under static stretch during prolonged sitting or lying. To investigate the effect of such chronic stimulation on adipocyte cell metabolism, we used an in vitro system to mimic the static stretch conditions. Under in vitro culture stretching, cells were analyzed at the single-cell level and we measured an increase in the projected area of the AD and higher content of lipid droplets. A decrease in the projected area of these cells' nucleus is associated with peroxisome proliferator-activated receptor-gamma expression and heterochromatin. This is the first study to reveal proteins that were altered under static stretch following a mass spectrometry analysis and main pathways that affect cell fate and metabolism. Bioinformatics analysis of the proteins indicated an increase in mitochondrial activity and associated pathways under static stretch stimulation. Quantification of the mitochondrial activity by 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT) assay and the ATPase related proteins specifically measured ATP5B indicated an increase in adipogenesis which points to a higher rate of cell metabolism under static stretch. In summary, our results elaborate on the metabolism of AD exposed to biomechanical stimulation, that is, associated with altered cellular protein profile and thereby influenced cell fate. The static stretch stimulation accelerated adipocyte differentiation through increased mitochondrial activity. Hence, in this study, we introduce a new perspective in understanding the molecular regulation of mechano-transduction in adipogenesis.

Mesenchymal Cell Growth and Differentiation on a New Biocomposite Material: A Promising Model for Regeneration Therapy.

Mesenchymal stem cells serve as the body's reservoir for healing and tissue regeneration. In cases of severe tissue trauma where there is also a need for tissue organization, a scaffold may be of use to support the cells in the damaged tissue. Such a scaffold should be composed of a material that can biomimic the mechanical and biological properties of the target tissues in order to support autologous cell-adhesion, their proliferation, and differentiation. In this study, we developed and assayed a new biocomposite made of unique collagen fibers and alginate hydrogel that was assessed for the ability to support mesenchymal cell-proliferation and differentiation. Analysis over 11 weeks in vitro demonstrated that the scaffold was biocompatible and supports the cells viability and differentiation to produce tissue-like structures or become adipocyte under differentiation medium. When the biocomposite was enriched with nano particles (NPs), mesenchymal cells grew well after uptake of fluorescein isothiocyanate (FITC) labeled NPs, maintained their viability, migrated through the biocomposite, reached, and adhered to the tissue culture dish. These promising findings revealed that the scaffold supports the growth and differentiation of mesenchymal cells that demonstrate their full physiological function with no sign of material toxicity. The cells' functionality performance indicates and suggests that the scaffold is suitable to be developed as a new medical device that has the potential to support regeneration and the production of functional tissue.

Adipocytes Migration is Altered Through Differentiation.

Adipogenesis is a developmental process in which an elongated preadipocyte differentiates to a round adipocyte along with the accumulation of lipid droplets. In the present study, we focus on the study of cell motility at the single-cell level, toward expanding our knowledge regarding the cytoskeleton alteration during differentiation; since-cell motility is mediated by cytoskeletal components. We used the holographic-microscopy live imaging technique to evaluate, for the first time in the literature, differences between the motility of nondifferentiated preadipocytes and differentiated mature adipocytes in living cell cultures over time. We revealed that mean motility speed of preadipocytes was significantly higher (fourfold) than that of adipocytes, and that the movement of preadipocytes is less consistent and more extensive. Furthermore, we found that preadipocytes tend to migrate to farther distances, while mature adipocytes remain relatively close to their original location. The results presented here are in agreement with the fact that the cytoskeleton of adipocytes is altered during differentiation and similarly, points to the fact that the cell-sensing mechanisms are changing during differentiation. Our research paves the way to gain better insights of the differentiation process and its implications on larger scale systems in the context of obesity.

Towards intervertebral disc engineering: Bio-mimetics of form and function of the annulus fibrosus lamellae.

The aging western society is heavily afflicted with intervertebral disc (IVD) degeneration. Replacement or repair of the degenerated IVD with an artificial bio-mimetic construct is one of the challenges of future research due to its complex structure and unique biomechanical function. Herein, biocomposite laminates made of long collagen fibers in unidirectional (-1.3 ±â€¯2.1°) and angle-plied ±â€¯30° orientations (30.4 ±â€¯6.4 and -29.8 ±â€¯4.5), embedded in alginate hydrogel, were fabricated to mimic the form of single annulus fibrosus (AF) lamella and the circumferential AF, respectively. The mechanical behavior of the composites was measured and compared with in vitro existing data of the human native AF as well as with new data obtained from ovine and bovine specimens. The mechanical behavior was found to reproduce the full stress- strain behavior of the human AF single lamella in several regions of the AF and the Young's modulus was 28.3 ±â€¯8.6 MPa. Moreover, the modulus of the angle-plied laminates was 16.8 ±â€¯2.9 MPa, which is approximately 5% less than the in vitro data. The full stress-strain behavior was also compared with bovine and ovine circumferential AF samples and found to be very similar, with a difference in the modulus of 4.1% and 19.7%, respectively. Moreover, an FE model of the L3-L4 functional spinal unit (FSU) was developed and calibrated to evaluate the mechanical ability of the biocomposite to be used as an AF substitute under physiological IVD loading modes. The biocomposite demonstrated a good ability to mimic the stiffness of the native tissue under physiologic loading modes as flexion, extension, lateral bending and compression, but was too flexible under torsion. It was found that the proposed biomimetics AF design resulted in a compatible function in several mechanical levels, which holds great potential to be used as a viable AF replacement towards full IVD engineering.

Molecular profile of ultrastructure changes of the ligamentum flavum related to lumbar spinal canal stenosis.

Lumbar spinal canal stenosis (LSCS) is a degenerative disease observed by hypertrophy of the ligamentum flavum (LF) that cause compression of the lumbar neural content. Diabetes mellitus (DM) is a risk factor for the disease and we have shown previously that DM increases the fibrosis and elastic fiber loss in patients with LSCS. The purpose of this study was to find the proteins that play a role in the development of this clinical pathogenesis and the effect of DM on protein expression. LF tissue retrieved from patients diagnosed with LSCS, some were also diagnosed with DM, were compared with LF from patients diagnosed with herniated nucleus pulposus (HNP). The tissues were analyzed by mass spectrometry for proteins profile alteration. We found that LF of LSCS/DM patients exhibited significantly higher levels of proteoglycan proteins and latent transforming growth factor ß-binding protein (LTBP2 and LTBP4). Additionally, an increase of HTRA serine protease 1 and insulin-like growth factor binding protein-5 were noted. The higher fibrosis was also associated with proteins related to inflammation and slower tissue repair. Collagen 6 and transforming growth factor inhibitor are related to activation of the anti-inflammatory M2 pathway that is associated with tissue repair. The decrease of these proteins expression in LSCS/DM is associated with increased levels and activation of M1 pro-inflammatory pathways. Interestingly, C3 and C4b members of the complement complex and mannose receptor-like protein (CLEC18) paralogous proteins were detectable solely at the LSCS/DM patients' samples. Histology analysis shows that inflammatory was induced by the hyperglycemic conditions in diabetic patients involve in altering the matrix compositions. Thus, the protein profiles associated with inflammatory pathways affecting the LF suggested increasing susceptibility of developing the degeneration under hyperglycemic conditions.

Noninvasive Continuous Monitoring of Adipocyte Differentiation: From Macro to Micro Scales.

3T3-L1 cells serve as model systems for studying adipogenesis and research of adipose tissue-related diseases, e.g. obesity and diabetes. Here, we present two novel and complementary nondestructive methods for adipogenesis analysis of living cells which facilitate continuous monitoring of the same culture over extended periods of time, and are applied in parallel at the macro- and micro-scales. At the macro-scale, we developed visual differences mapping (VDM), a novel method which allows to determine level of adipogenesis (LOA)-a numerical index which quantitatively describes the extent of differentiation in the whole culture, and percentage area populated by adipocytes (PAPBA) across a whole culture, based on the apparent morphological differences between preadipocytes and adipocytes. At the micro-scale, we developed an improved version of our previously published image-processing algorithm, which now provides data regarding single-cell morphology and lipid contents. Both methods were applied here synergistically for measuring differentiation levels in cultures over multiple weeks. VDM revealed that the mean LOA value reached 1.11 ± 0.06 and the mean PAPBA value reached >60%. Micro-scale analysis revealed that during differentiation, the cells transformed from a fibroblast-like shape to a circular shape with a build-up of lipid droplets. We predict a vast potential for implementation of these methods in adipose-related pharmacological research, such as in metabolic-syndrome studies.

How is mechanobiology involved in mesenchymal stem cell differentiation toward the osteoblastic or adipogenic fate?

Mechanobiology plays a major role in transducing physical cues from the dynamic cellular environment into biochemical modifications that promote cell-specific differentiation paths. Mesenchymal stem cells in the bone marrow or in other mesenchymal tissues will differentiate according to the expression of transcription factors (TFs) that govern their lineage commitment. The favoring of either osteogenic or adipogenic differentiation relies on TF expression as well as mechanical properties of the cells' niche that are translated into the activation of certain signaling pathways. Physical factors can induce significant shifts in bipotential lineage commitment between osteogenesis and adipogenesis. The stiffness of the extracellular matrix (ECM) surrounding a cell, varying greatly from rigid environments close to the bone surface to softer regions in the bone marrow, can influence the path of differentiation. Additionally, mechanical loading through exercise appears to favor osteogenesis whereas disuse conditions seem to promote adipogenesis.

Cell shape alteration during adipogenesis is associated with coordinated matrix cues.

Obesity has become one of the leading pathophysiologic disorders in recent years. Adipose tissue is the main tissue related to obesity and is known to play a role in various physiological complications, including type 2 diabetes. To better understand how the fat tissue develops, we used an in vitro live cell imaging system to quantify the adipogenesis by means of nondestructive digital imaging to monitor the accumulation of intracellular lipid droplets (LDs), a hallmark of adipogenesis, from the macro- to the micro-scale. Analyzing the cells' shape at the single-cell level allows to quantify the cells' shape change from a fibroblast to spherical morphology, indicating the start of adipogenesis. To reveal the molecular alterations, we applied a proteomic approach using high-resolution mass spectrometry of the proliferation, confluent fibroblasts and of adipocytes. During this process, we noted the reorganization of the cells' extracellular matrix (ECM) network microenvironment from fibrillary collagen types I, III and V to collagens IV and VI, which affected the cells niche. The changes in ECM are translated for cytoskeleton remodeling according to cell fate-determining mechanisms. We quantified the cytoskeleton rearrangement of long oriented actin fibers or short cortical and disorganized fibers, associated with LDs accumulation in adipocytes. Developing in vitro models and analytical methods enable us to study differentiation into adipocytes that will advance our understanding regarding the niche conditions that affect adipogenesis. Consequently, this will enable the development of new modalities to prevent obesity and its deleterious outcomes and to develop potential treatments to battle pathophysiology-related diseases.

RGD-modified dihydrolipoamide dehydrogenase as a molecular bridge for enhancing the adhesion of bone forming cells to titanium dioxide implant surfaces.

Titanium and its alloys are widely used in dental- and orthopedic implants, the outer surface of which is often oxidized to titanium dioxide (TiO2 ). To achieve efficient osseointegration with bone-forming cells, it is desirable to counter the formation of the soft fibrous tissue around the implant by creating strong and stable interactions between the implant surface and bone-forming osteoblasts. To address this challenge, a bioactive coating had to be designed. Protein adsorption to TiO2 is well known in the literature, but it is mostly characterized by weak associations, rendering less efficient implant osseointegration. We have previously demonstrated the unique conjugation between the dihydrolipoamide dehydrogenase (DLDH) protein and TiO2 surfaces, based on specific coordinative bonding via Cys-His-Glu-Asp motif residues. To enhance cell binding to DLDH and facilitate osseointegration, DLDH was bioengineered to include Arg-Gly-Asp (RGD) moieties (DLDHRGD ). Coating TiO2 disks with DLDHRGD led to improved adherence of integrin-expressing osteogenic MBA-15 to the surface of the disks. Following the enhanced adsorption, higher proliferation rates of the adherent cells, as well as faster mineralization were observed, compared to controls. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 107A: 545-551, 2019.