Hydroxyapatite/alginate/gellan gum inks with osteoconduction and osteogenic potential for bioprinting bone tissue analogues.

There is a growing demand for engineered bone tissues custom-designed to match the patient-specific defect size and in vitro models for studying bone diseases and/or drug screening. Herein, we propose a bioprinted bone tissue construct using SaOs-2 cells within alginate/gellan gum/hydroxyapatite inks. Different ink formulations were developed with varying hydroxyapatite content and then evaluated for viscoelasticity, printability, biomineralization properties, post-printing viability, proliferation, metabolic activity, and osteogenic phenotype of SaOs-2-encapsulated cells. Results indicate that ink formulations exhibit non-Newtonian shear-thinning behaviour, maintaining shape integrity and structural stability post-printing. Ink mineralization rates increase with the hydroxyapatite content, rendering them suitable for bone defect strategies. Post-printed cells in the developed constructs remain live, spreading, and metabolically active but do not proliferate. Osteogenic gene and protein expression, both early and late, show upregulation at day 7 relative to day 1, followed by downregulation at day 14. Lower hydroxyapatite content inks demonstrate up to fourfold upregulation in genes and proteins at most time points. Additionally, these constructs release calcium and phosphate at levels conducive to mineralization. Overall, the tissue-engineered miniaturized constructs not only meet the criteria for early-stage bone defect/fracture regeneration but also serve as a promising platform for drug screening and evaluating potential therapeutic treatments.

Development of Conjugated Kefiran-Chondroitin Sulphate Cryogels with Enhanced Properties for Biomedical Applications.

Hydrogels based on natural polysaccharides can have unique properties and be tailored for several applications, which may be mainly limited by the fragile structure and weak mechanical properties of this type of system. We successfully prepared cryogels made of newly synthesized kefiran exopolysaccharide-chondroitin sulfate (CS) conjugate via carbodiimide-mediated coupling to overcome these drawbacks. The freeze-thawing procedure of cryogel preparation followed by lyophilization is a promising route to fabricate polymer-based scaffolds with countless and valuable biomedical applications. The novel graft macromolecular compound (kefiran-CS conjugate) was characterized through 1H-NMR and FTIR spectroscopy-which confirmed the structure of the conjugate, differential scanning calorimetry (DSC) and thermogravimetric analysis (TGA)-which mirrored good thermal stability (degradation temperature of about 215 °C) and, finally, gel permeation chromatography-size exclusion chromatography (GPC-SEC)-which proved an increased molecular weight due to chemical coupling of kefiran with CS. At the same time, the corresponding cryogels physically crosslinked after the freeze-thawing procedure were investigated by scanning electron microscopy (SEM), Micro-CT, and dynamic rheology. The results revealed a prevalent contribution of elastic/storage component to the viscoelastic behavior of cryogels in swollen state, a micromorphology with micrometer-sized open pores fully interconnected, and high porosity (ca. 90%) observed for freeze-dried cryogels. Furthermore, the metabolic activity and proliferation of human adipose stem cells (hASCs), when cultured onto the developed kefiran-CS cryogel, was maintained at a satisfactory level over 72 h. Based on the results obtained, it can be inferred that the newly freeze-dried kefiran-CS cryogels possess a host of unique properties that render them highly suitable for use in tissue engineering, regenerative medicine, drug delivery, and other biomedical applications where robust mechanical properties and biocompatibility are crucial.

Glial Cell Line-Derived Neurotrophic Factor-Loaded CMCht/PAMAM Dendrimer Nanoparticles for Peripheral Nerve Repair.

(1) Background: Peripheral nerve injuries represent a major clinical challenge. If nerve ends retract, there is no spontaneous regeneration and grafts are required to proximate the nerve ends and give continuity to the nerve. (2) Methods: GDNF-loaded NPs were characterized physicochemically. For that, NPs stability at different pH's was assessed, and GDNF release was studied through ELISA. In vitro studies are performed with Schwann cells, and the NPs are labeled with fluorescein-5(6)-isothiocyanate for uptake experiments with SH-SY5Y neural cells. (3) Results: GDNF-loaded NPs are stable in physiological conditions, releasing GDNF in a two-step profile, which is beneficial for nerve repair. Cell viability is improved after 1 day of culture, and the uptake is near 99.97% after 3 days of incubation. (4) Conclusions: The present work shows the efficiency of using CMCht/PAMAM NPs as a GDNF-release system to act on peripheral nerve regeneration.

Recent approaches towards bone tissue engineering.

Bone tissue engineering approaches have evolved towards addressing the challenges of tissue mimetic requirements over the years. Different strategies have been combining scaffolds, cells, and biologically active cues using a wide range of fabrication techniques, envisioning the mimicry of bone tissue. On the one hand, biomimetic scaffold-based strategies have been pursuing different biomaterials to produce scaffolds, combining with diverse and innovative fabrication strategies to mimic bone tissue better, surpassing bone grafts. On the other hand, biomimetic scaffold-free approaches mainly foresee replicating endochondral ossification, replacing hyaline cartilage with new bone. Finally, since bone tissue is highly vascularized, new strategies focused on developing pre-vascularized scaffolds or pre-vascularized cellular aggregates have been a motif of study. The recent biomimetic scaffold-based and scaffold-free approaches in bone tissue engineering, focusing on materials and fabrication methods used, are overviewed herein. The biomimetic vascularized approaches are also discussed, namely the development of pre-vascularized scaffolds and pre-vascularized cellular aggregates.

Carbon nanotube-reinforced cell-derived matrix-silk fibroin hierarchical scaffolds for bone tissue engineering applications.

In bone tissue engineering, the development of advanced biomimetic scaffolds has led to the quest for biomotifs in scaffold design that better recreate the bone matrix structure and composition and hierarchy at different length scales. In this study, an advanced hierarchical scaffold consisting of silk fibroin combined with a decellularized cell-derived extracellular matrix and reinforced with carbon nanotubes was developed. The goal of the carbon nanotube-reinforced cell-derived matrix-silk fibroin hierarchical scaffolds is to harvest the individual properties of their constituents to introduce hierarchical capacity in order to improve standard silk fibroin scaffolds. The scaffolds were fabricated using enzymatic cross-linking, freeze modeling, and decellularization methods. The developed scaffolds were assessed for the pore structure and mechanical properties showing satisfying results to be used in bone regeneration. The developed carbon nanotube-reinforced cell-derived matrix-silk fibroin hierarchical scaffolds were shown to be bioactive in vitro and expressed no hemolytic effect. Furthermore, cellular in vitro studies on human adipose-derived stem cells (hASCs) showed that scaffolds supported cell proliferation. The hASCs seeded onto these scaffolds evidenced similar metabolic activity to standard silk fibroin scaffolds but increased ALP activity. The histological staining showed cell infiltration into the scaffolds and visible collagen production. The expression of several osteogenic markers was investigated, further supporting the osteogenic potential of the developed carbon nanotube-reinforced cell-derived matrix-silk fibroin hierarchical scaffolds. The hemolytic assay demonstrated the hemocompatibility of the hierarchical scaffolds. Overall, the carbon nanotube-reinforced cell-derived matrix-silk fibroin hierarchical scaffolds presented the required architecture for bone tissue engineering applications.

Combining experiments and in silico modeling to infer the role of adhesion and proliferation on the collective dynamics of cells.

The collective dynamics of cells on surfaces and interfaces poses technological and theoretical challenges in the study of morphogenesis, tissue engineering, and cancer. Different mechanisms are at play, including, cell-cell adhesion, cell motility, and proliferation. However, the relative importance of each one is elusive. Here, experiments with a culture of glioblastoma multiforme cells on a substrate are combined with in silico modeling to infer the rate of each mechanism. By parametrizing these rates, the time-dependence of the spatial correlation observed experimentally is reproduced. The obtained results suggest a reduction in cell-cell adhesion with the density of cells. The reason for such reduction and possible implications for the collective dynamics of cancer cells are discussed.

Microfluidic Devices and Three Dimensional-Printing Strategies for in vitro Models of Bone.

Bone is a complex and highly dynamic tissue, which has been worldwide studied, from fundamental biology to tissue engineering fields. Even so, current in vitro models do not truly replicate the native bone tissue environment. For so, new and improved in vitro tissue models are necessary to obtain more reliable data, not only in a development point of view, but also to fasten the translation of new drugs into the clinics. In this reasoning, tissue-engineering strategies were applied to develop mimetic and three-dimensional (3D) microenvironments, which were associated with microfluidic devices for the development of more complex and realistic systems. Such devices mimic blood vessels that are present in the native tissue, thus enabling the study of complex biological mechanism as such as bone angiogenesis. More recently, 3D printing has been pursued to produce more intricate microfluidic devices and engineered tissues in a single step. The ability to print spatially controlled structures composed of different biomaterials, growth factors and cells caught the attention of scientists for the development of more efficient in vitro models. Additionally, it allows obtaining microfluidic devices and/or engineered tissues with the desired architecture within a small amount of time and with reduced costs. Recently, the use of high-resolution scanning boosted the production of patient-specific implants. Despite the difficulties associated with 3D printed structures that still need to be overcome, it has been proven to be a valuable tool to accomplish a new generation of 3D bioprinted bone-on-a-chip platforms.

Nanoparticles and Microfluidic Devices in Cancer Research.

Cancer is considered the disease of the century, which can be easily understood considering its increasing incidence worldwide. Over the last years, nanotechnology has been presenting promising theranostic approaches to tackle cancer, as the development of nanoparticle-based therapies. But, regardless of the promising outcomes within in vitro settings, its translation into the clinics has been delayed. One of the main reasons is the lack of an appropriate in vitro model, capable to mimic the true environment of the human body, to test the designed nanoparticles. In fact, most of in vitro models used for the validation of nanoparticle-based therapies do not address adequately the complex barriers that naturally occur in a tumor scenario, as such as blood vessels, the interstitial fluid pressure or the interactions with surrounding cells that can hamper the proper delivery of the nanoparticles into the desired site. In this reasoning, to get a step closer to the in vivo reality, it has been proposed of the use of microfluidic devices. In fact, microfluidic devices can be designed on-demand to exhibit complex structures that mimic tissue/organ-level physiological architectures. Even so, despite microfluidic-based in vitro models do not compare with the reality and complexity of the human body, the most complex systems created up to now have been showing similar results to in vivo animal models. Microfluidic devices have been proven to be a valuable tool to accomplish more realistic tumour's environment. The recent advances in this field, and in particular, the ones enabling the rapid test of new therapies, and show great promise to be translated to the clinics will be overviewed herein.

Finding the perfect match between nanoparticles and microfluidics to respond to cancer challenges.

The clinical translation of new cancer theranostic has been delayed by inherent cancer's heterogeneity. Additionally, this delay has been enhanced by the lack of an appropriate in vitro model, capable to produce accurate data. Nanoparticles and microfluidic devices have been used to obtain new and more efficient strategies to tackle cancer challenges. On one hand, nanoparticles-based therapeutics can be modified to target specific cells, and/or molecules, and/or modified with drugs, releasing them over time. On the other hand, microfluidic devices allow the exhibition of physiologically complex systems, incorporation of controlled flow, and control of the chemical environment. Herein, we review the use of nanoparticles and microfluidic devices to address different cancer challenges, such as detection of CTCs and biomarkers, point-of-care devices for early diagnosis and improvement of therapies. The future perspectives of cancer challenges are also addressed herein.

Lactoferrin-Hydroxyapatite Containing Spongy-Like Hydrogels for Bone Tissue Engineering.

The development of bioactive and cell-responsive materials has fastened the field of bone tissue engineering. Gellan gum (GG) spongy-like hydrogels present high attractive properties for the tissue engineering field, especially due to their wide microarchitecture and tunable mechanical properties, as well as their ability to entrap the responsive cells. Lactoferrin (Lf) and Hydroxyapatite (HAp) are bioactive factors that are known to potentiate faster bone regeneration. Thus, we developed an advanced three-dimensional (3D) biomaterial by integrating these bioactive factors within GG spongy-like hydrogels. Lf-HAp spongy-like hydrogels were characterized in terms of microstructure, water uptake, degradation, and concomitant release of Lf along the time. Human adipose-derived stem cells (hASCs) were seeded and the capacity of these materials to support hASCs in culture for 21 days was assessed. Lf addition within GG spongy-like hydrogels did not change the main features of GG spongy-like hydrogels in terms of porosity, pore size, degradation, and water uptake commitment. Nevertheless, HAp addition promoted an increase of the pore wall thickness (from ~13 to 28 µm) and a decrease on porosity (from ~87% to 64%) and mean pore size (from ~12 to 20 µm), as well as on the degradability and water retention capabilities. A sustained release of Lf was observed for all the formulations up to 30 days. Cell viability assays showed that hASCs were viable during the culture period regarding cell-laden spongy-like hydrogels. Altogether, we demonstrate that GG spongy-like hydrogels containing HAp and Lf in high concentrations gathered favorable 3D bone-like microenvironment with an increased hASCs viability with the presented results.

Scaffolding Strategies for Tissue Engineering and Regenerative Medicine Applications.

During the past two decades, tissue engineering and the regenerative medicine field have invested in the regeneration and reconstruction of pathologically altered tissues, such as cartilage, bone, skin, heart valves, nerves and tendons, and many others. The 3D structured scaffolds and hydrogels alone or combined with bioactive molecules or genes and cells are able to guide the development of functional engineered tissues, and provide mechanical support during in vivo implantation. Naturally derived and synthetic polymers, bioresorbable inorganic materials, and respective hybrids, and decellularized tissue have been considered as scaffolding biomaterials, owing to their boosted structural, mechanical, and biological properties. A diversity of biomaterials, current treatment strategies, and emergent technologies used for 3D scaffolds and hydrogel processing, and the tissue-specific considerations for scaffolding for Tissue engineering (TE) purposes are herein highlighted and discussed in depth. The newest procedures focusing on the 3D behavior and multi-cellular interactions of native tissues for further use for in vitro model processing are also outlined. Completed and ongoing preclinical research trials for TE applications using scaffolds and hydrogels, challenges, and future prospects of research in the regenerative medicine field are also presented.

Biological performance of a promising Kefiran-biopolymer with potential in regenerative medicine applications: a comparative study with hyaluronic acid.

Kefiran from kefir grains, an exopolysaccharide (EPS) produced by lactic acid bacteria (LAB), has received an increasing interest because of its safe status. This natural biopolymer is a water-soluble glucogalactan with probed health-promoting properties. However, its biological performance has yet to be completely recognized and properly exploited. This research was carried out to evaluate the in vitro antioxidant and the in vitro anti-inflammatory properties of Kefiran biopolymer. Regarding antioxidant activity, the results demonstrated that the Kefiran extract possessed the strongest reducing power and superoxide radical scavenging, over hyaluronic acid (HA, gold standard viscosupplementation treatment). This exopolysaccharide showed a distinct antioxidant performance in the majority of in vitro working mechanisms of antioxidant activity comparing to HA. Moreover, Kefiran presented an interesting capacity to scavenge nitric oxide radical comparing to the gold standard that did not present any potency. Finally, the cytotoxic effects of Kefiran extracts on hASCs were also performed and demonstrated no cytotoxic response, ability to improve cellular function of hASCs. This study demonstrated that Kefiran represented a great scavenger for reactive oxygen and nitrogen species and showed also that it could be an excellent candidate to promote tissue repair and regeneration.

Tissue Engineering Strategies for Osteochondral Repair.

Tissue engineering strategies have been pushing forward several fields in the range of biomedical research. The musculoskeletal field is not an exception. In fact, tissue engineering has been a great asset in the development of new treatments for osteochondral lesions. Herein, we overview the recent developments in osteochondral tissue engineering. Currently, the treatments applied in a clinical scenario have shown some drawbacks given the difficulty in regenerating a fully functional hyaline cartilage. Among the different strategies designed for osteochondral regeneration, it is possible to identify cell-free strategies, scaffold-free strategies, and advanced strategies, where different materials are combined with cells. Cell-free strategies consist in the development of scaffolds in the attempt to better fulfill the requirements of the cartilage regeneration process. For that, different structures have been designed, from monolayers to multilayered structures, with the intent to mimic the osteochondral architecture. In the case of scaffold-free strategies, they took advantage on the extracellular matrix produced by cells. The last strategy relies in the development of new biomaterials capable of mimicking the extracellular matrix. This way, the cell growth, proliferation, and differentiation at the lesion site are expedited, exploiting the self-regenerative potential of cells and its interaction with biomolecules. Overall, despite the difficulties associated with each approach, tissue engineering has been proven a valuable tool in the regeneration of osteochondral lesions and together with the latest advances in the field, promises to revolutionize personalized therapies.

Combinatory approach for developing silk fibroin scaffolds for cartilage regeneration.

Several processing technologies and engineering strategies have been combined to create scaffolds with superior performance for efficient tissue regeneration. Cartilage tissue is a good example of that, presenting limited self-healing capacity together with a high elasticity and load-bearing properties. In this work, novel porous silk fibroin (SF) scaffolds derived from horseradish peroxidase (HRP)-mediated crosslinking of highly concentrated aqueous SF solution (16 wt%) in combination with salt-leaching and freeze-drying methodologies were developed for articular cartilage tissue engineering (TE) applications. The HRP-crosslinked SF scaffolds presented high porosity (89.3 ±â€¯0.6%), wide pore distribution and high interconnectivity (95.9 ±â€¯0.8%). Moreover, a large swelling capacity and favorable degradation rate were observed up to 30 days, maintaining the porous-like structure and ß-sheet conformational integrity obtained with salt-leaching and freeze-drying processing. The in vitro studies supported human adipose-derived stem cells (hASCs) adhesion, proliferation, and high glycosaminoglycans (GAGs) synthesis under chondrogenic culture conditions. Furthermore, the chondrogenic differentiation of hASCs was assessed by the expression of chondrogenic-related markers (collagen type II, Sox-9 and Aggrecan) and deposition of cartilage-specific extracellular matrix for up to 28 days. The cartilage engineered constructs also presented structural integrity as their mechanical properties were improved after chondrogenic culturing. Subcutaneous implantation of the scaffolds in CD-1 mice demonstrated no necrosis or calcification, and deeply tissue ingrowth. Collectively, the structural properties and biological performance of these porous HRP-crosslinked SF scaffolds make them promising candidates for cartilage regeneration. STATEMENT OF SIGNIFICANCE: In cartilage tissue engineering (TE), several processing technologies have been combined to create scaffolds for efficient tissue repair. In our study, we propose novel silk fibroin (SF) scaffolds derived from enzymatically crosslinked SF hydrogels processed by salt-leaching and freeze-drying technologies, for articular cartilage applications. Though these scaffolds, we were able to combine the elastic properties of hydrogel-based systems, with the stability, resilience and controlled porosity of scaffolds processed via salt-leaching and freeze-drying technologies. SF protein has been extensively explored for TE applications, as a result of its mechanical strength, elasticity, biocompatibility, and biodegradability. Thus, the structural, mechanical and biological performance of the proposed scaffolds potentiates their use as three-dimensional matrices for cartilage regeneration.

Differentiation of osteoclast precursors on gellan gum-based spongy-like hydrogels for bone tissue engineering.

Bone tissue engineering with cell-scaffold constructs has been attracting a lot of attention, in particular as a tool for the efficient guiding of new tissue formation. However, the majority of the current strategies used to evaluate novel biomaterials focus on osteoblasts and bone formation, while osteoclasts are often overlooked. Consequently, there is limited knowledge on the interaction between osteoclasts and biomaterials. In this study, the ability of spongy-like gellan gum and hydroxyapatite-reinforced gellan gum hydrogels to support osteoclastogenesis was investigated in vitro. First, the spongy-like gellan gum and hydroxyapatite-reinforced gellan gum hydrogels were characterized in terms of microstructure, water uptake and mechanical properties. Then, bone marrow cells isolated from the long bones of mice and cultured in spongy-like hydrogels were treated with 1,25-dihydroxyvitamin D3 to promote osteoclastogenesis. It was shown that the addition of HAp to spongy-like gellan gum hydrogels enables the formation of larger pores and thicker walls, promoting an increase in stiffness. Hydroxyapatite-reinforced spongy-like gellan gum hydrogels support the formation of the aggregates of tartrate-resistant acid phosphatase-stained cells and the expression of genes encoding DC-STAMP and Cathepsin K, suggesting the differentiation of bone marrow cells into pre-osteoclasts. The hydroxyapatite-reinforced spongy-like gellan gum hydrogels developed in this work show promise for future use in bone tissue scaffolding applications.

A semiautomated microfluidic platform for real-time investigation of nanoparticles' cellular uptake and cancer cells' tracking.

AIM: Develop a platform composed of labeled dendrimer nanoparticles (NPs) and a microfluidic device for real-time monitoring of cancer cells fate. MATERIALS & METHODS: Carboxymethylchitosan/poly(amidoamine) dendrimer NPs were labeled with fluorescein-5(6)-isothiocyanate and characterized using different physicochemical techniques. After, HeLa, HCT-116 and U87MG were cultured in the presence of NPs, and cell viability and internalization efficiency in static (standard culture) and dynamic (microfluidic culture) conditions were investigated. RESULTS: Cancer cells cultured with NPs in dynamic conditions were viable and presented higher internalization levels as compared with static 2D cultures. CONCLUSION: This work demonstrated that the proposed microfluidic-based platform allows real-time monitoring, which upon more studies, namely, the assessment of an anticancer drug release effect could be used for cancer theranostics.

Hydrogel depots for local co-delivery of osteoinductive peptides and mesenchymal stem cells.

The outcome of cell-based therapies can benefit from carefully designed cell carriers. A multifunctional injectable vehicle for the co-delivery of human mesenchymal stem cells (hMSCs) and osteoinductive peptides is proposed, to specifically direct hMSCs osteogenic differentiation. The osteogenic growth peptide (OGP) inspired the design of two peptides, where the bioactive portion of OGP was flanked by a protease-sensitive linker, or its scrambled sequence, to provide faster and slower release rates, respectively. Peptides were fully characterized and chemically grafted to alginate. Both OGP analogs released bioactive fragments in vitro, at different kinetics, which stimulated hMSCs proliferation and osteogenesis. hMSCs-laden OGP-alginate hydrogels were tested at an ectopic site in a xenograft mouse model. After 4weeks, OGP-alginate hydrogels were more degraded and colonized by vascularized connective tissue than the control (without OGP). hMSCs were able to proliferate, migrate outward the hydrogels, produce endogenous extracellular matrix and mineralize it. Moreover, OGP-groups stimulated hMSCs osteogenesis, as compared with the control. Overall, the ability of the proposed platform to direct the fate of transplanted hMSCs in loco was demonstrated, and OGP-releasing hydrogels emerged as a potentially useful system to promote bone regeneration.

Effect of cell density on mesenchymal stem cells aggregation in RGD-alginate 3D matrices under osteoinductive conditions.

Cellular activities in 3D are differentially affected by several matrix-intrinsic and extrinsic factors. This study highlights the relevance of optimizing initial cell densities when establishing 3D cultures for specific applications. Independently of the entrapping density, MSCs cultured within RGD-alginate hydrogels showed steady-state levels of metabolic activity and were in a nearly non-proliferative state, but recovered "normal" activity levels when retrieved from 3D matrices and re-cultured as monolayers. Importantly, high-densities promoted the establishment of cell-cell contacts with formation of multicellular clusters stabilized by endogenous ECM, and also stimulated MSCs osteogenic differentiation. These MSC-ECM microtissues may be used as building blocks for tissue engineering.

Matrix-driven formation of mesenchymal stem cell-extracellular matrix microtissues on soft alginate hydrogels.

Mesenchymal stem cells (MSCs) can be made to rearrange into microtissues in response to specific matrix cues, a process that depends on a balance between cell-matrix and cell-cell interactions. The effect of such cues, and especially their interplay, is still not fully understood, particularly in three-dimensional (3-D) systems. Here, the behaviour of human MSCs cultured within hydrogel matrices with tailored stiffness and composition was evaluated. MSC aggregation occurred only in more compliant matrices (G'≤ 120 Pa), when compared to stiffer ones, both in the presence and in the absence of matrix-bound arginine-glycine-aspartic acid cell-adhesion ligands (RGD; 0, 100 and 200 µM). Fibronectin assembly stabilized cell-cell contacts within aggregates, even in non-adhesive matrices. However, MSCs were able to substantially contract the artificial matrix only when RGD was present. Moreover, compliant matrices facilitated cell proliferation and provided an environment conducive for MSC osteogenic differentiation, even without RGD. Cell interactions with the original matrix became less important as time progressed, while the de novo-produced extracellular matrix became a more critical determinant of cell fate. These data provide further insights into the mechanisms by which MSCs sense their microenvironment to organize into tissues, and provide new clues to the design of cell-instructive 3-D matrices.

Functionalization of biomaterials with small osteoinductive moieties.

Human mesenchymal stem cells (MSCs) are currently recognized as a powerful cell source for regenerative medicine, notably for their capacity to differentiate into multiple cell types. The combination of MSCs with biomaterials functionalized with instructive cues can be used as a strategy to direct specific lineage commitment, and can thus improve the therapeutic efficacy of these cells. In terms of biomaterial design, one common approach is the functionalization of materials with ligands capable of directly binding to cell receptors and trigger specific differentiation signaling pathways. Other strategies focus on the use of moieties that have an indirect effect, acting, for example, as sequesters of bioactive ligands present in the extracellular milieu that, in turn, will interact with cells. Compared with complex biomolecules, the use of simple compounds, such as chemical moieties and peptides, and other small molecules can be advantageous by leading to less expensive and easily tunable biomaterial formulations. This review describes different strategies that have been used to promote substrate-mediated guidance of osteogenic differentiation of immature osteoblasts, osteoprogenitors and MSCs, through chemically conjugated small moieties, both in two- and three-dimensional set-ups. In each case, the selected moiety, the coupling strategy and the main findings of the study were highlighted. The latest advances and future perspectives in the field are also discussed.