Platelet-Derived Extracellular Vesicles Promote Tenogenic Differentiation of Stem Cells on Bioengineered Living Fibers.

Tendon mimetic scaffolds that recreate the tendon hierarchical structure and niche have increasing potential to fully restore tendon functionality. However, most scaffolds lack biofunctionality to boost the tenogenic differentiation of stem cells. In this study, we assessed the role of platelet-derived extracellular vesicles (EVs) in stem cells' tenogenic commitment using a 3D bioengineered in vitro tendon model. First, we relied on fibrous scaffolds coated with collagen hydrogels encapsulating human adipose-derived stem cells (hASCs) to bioengineer our composite living fibers. We found that the hASCs in our fibers showed high elongation and cytoskeleton anisotropic organization, typical of tenocytes. Moreover, acting as biological cues, platelet-derived EVs boosted the hASCs' tenogenic commitment, prevented phenotypic drift, enhanced the deposition of the tendon-like extracellular matrix, and induced lower collagen matrix contraction. In conclusion, our living fibers provided an in vitro system for tendon tissue engineering, allowing us to study not only the tendon microenvironment but also the influence of biochemical cues on stem cell behavior. More importantly, we showed that platelet-derived EVs are a promising biochemical tool for tissue engineering and regenerative medicine applications that are worthy of further exploration, as paracrine signaling might potentiate tendon repair and regeneration.

Therapeutic Effects of Platelet-Derived Extracellular Vesicles in a Bioengineered Tendon Disease Model.

Tendon injuries represent over 30-50% of musculoskeletal disorders worldwide, yet the available therapies do not provide complete tendon repair/regeneration and full functionality restoring. Extracellular vesicles (EVs), membrane-enclosed nanoparticles, have emerged as the next breakthrough in tissue engineering and regenerative medicine to promote endogenous tissue regeneration. Here, we developed a 3D human in vitro model mimicking the signature of pathological tendon and used it to evaluate the influence that different platelet-derived EVs might have in tendon tissue repair mechanisms. For this, different EV populations isolated from platelets, small EVs (sEVs) and medium EVs (mEVs), were added to the culture media of human tendon-derived cells (hTDCs) cultured on isotropic nanofibrous scaffolds. The platelet-derived EVs increased the expression of tenogenic markers, promoted a healthy extracellular matrix (ECM) remodeling, and the synthesis of anti-inflammatory mediators. These findings suggest that platelet EVs provided relevant biochemical cues that potentiated a recovery of hTDCs phenotype from a diseased to a healthy state. Thus, this study opens new perspectives for the translation of platelet-derived EVs as therapeutics.

Human Platelet Lysate-Loaded Poly(ethylene glycol) Hydrogels Induce Stem Cell Chemotaxis In Vitro.

Platelet lysates (PL) contain a selection of proteins and growth factors (GFs) that are known to mediate cell activity. Many of these biomolecules have been identified as chemoattractants with the capacity to induce cell migration. In order to effectively deliver and retain these biomolecules to the site of injury, a scaffold containing PL could be an option. We use poly(ethylene glycol) (PEG) hydrogels consisting of 90 vol % PL to investigate their migratory potential on human mesenchymal stem cells (hMSCs). Cells exposed to these hydrogels were tracked, resulting in cell trajectories and detailed migratory parameters (velocity, Euclidean distance, directness, and forward migration index). Volumetric swelling ratios, hydrogel mechanical properties, and the release kinetics of proteins and GFs from hydrogels were also assessed. Furthermore, hMSC spheroids were encapsulated within the hydrogels to qualitatively assess cell invasion by means of sprouting and disintegration of the spheroid. Cell spheroids encapsulated within the PL-PEG gels exhibited initial outgrowths and eventually colonized the 3D matrix successfully. Results from this study confirmed that hMSCs exhibit directional migration toward the PL-loaded hydrogel with increased velocity and directness, compared to the controls. Overall, the incorporation of PL renders the PEG hydrogel bioactive. This study demonstrates the capacity of PL-loaded hydrogel constructs to attract stem cells for endogenous tissue engineering purposes.

Intrinsically Bioactive Cryogels Based on Platelet Lysate Nanocomposites for Hemostasis Applications.

The currently used hemostatic agents are highly effective in stopping hemorrhages but have a limited role in the modulation of the wound-healing environment. Herein, we propose an intrinsically bioactive hemostatic cryogel based on platelet lysate (PL) and aldehyde-functionalized cellulose nanocrystals (a-CNCs). PL has attracted great attention as an inexpensive milieu of therapeutically relevant proteins; however, its application as a hemostatic agent exhibits serious constraints (e.g., structural integrity and short shelf-life). The incorporation of a-CNCs reinforced the low-strength PL matrix by covalent cross-linking its amine groups that exhibit an elastic interconnected porous network after full cryogelation. Upon blood immersion, the PL-CNC cryogels absorbed higher volumes of blood at a faster rate than commercial hemostatic porcine gelatin sponges. Simultaneously, the cryogels released biomolecules that increased stem cell proliferation, metabolic activity, and migration as well as downregulated the expression of markers of the fibrinolytic process. In an in vivo liver defect model, PL-CNC cryogels showed similar hemostatic performance in comparison with gelatin sponges and normal material-induced tissue response upon subcutaneous implantation. Overall, owing to their structure and bioactive composition, the proposed PL-CNC cryogels provide an alternative off-the-shelf hemostatic and antibacterial biomaterial with the potential to deliver therapeutically relevant proteins in situ.

Natural-Based Hydrogels for Tissue Engineering Applications.

In the field of tissue engineering and regenerative medicine, hydrogels are used as biomaterials to support cell attachment and promote tissue regeneration due to their unique biomimetic characteristics. The use of natural-origin materials significantly influenced the origin and progress of the field due to their ability to mimic the native tissues' extracellular matrix and biocompatibility. However, the majority of these natural materials failed to provide satisfactory cues to guide cell differentiation toward the formation of new tissues. In addition, the integration of technological advances, such as 3D printing, microfluidics and nanotechnology, in tissue engineering has obsoleted the first generation of natural-origin hydrogels. During the last decade, a new generation of hydrogels has emerged to meet the specific tissue necessities, to be used with state-of-the-art techniques and to capitalize the intrinsic characteristics of natural-based materials. In this review, we briefly examine important hydrogel crosslinking mechanisms. Then, the latest developments in engineering natural-based hydrogels are investigated and major applications in the field of tissue engineering and regenerative medicine are highlighted. Finally, the current limitations, future challenges and opportunities in this field are discussed to encourage realistic developments for the clinical translation of tissue engineering strategies.

Multifunctional magnetic-responsive hydrogels to engineer tendon-to-bone interface.

Photocrosslinkable magnetic hydrogels are attracting great interest for tissue engineering strategies due to their versatility and multifunctionality, including their remote controllability ex vivo, thus enabling engineering complex tissue interfaces. This study reports the development of a photocrosslinkable magnetic responsive hydrogel made of methacrylated chondroitin sulfate (MA-CS) enriched with platelet lysate (PL) with tunable features, envisioning their application in tendon-to-bone interface. MA-CS coated iron-based magnetic nanoparticles were incorporated to provide magnetic responsiveness to the hydrogel. Osteogenically differentiated adipose-derived stem cells and/or tendon-derived cells were encapsulated within the hydrogel, proliferating and expressing bone- and tendon-related markers. External magnetic field (EMF) application modulated the swelling, degradation and release of PL-derived growth factors, and impacted both cell morphology and the expression and synthesis of tendon- and bone-like matrix with a more evident effect in co-cultures. Overall, the developed magnetic responsive hydrogel represents a potential cell carrier system for interfacial tissue engineering with EMF-controlled properties.

3D Mimicry of Native-Tissue-Fiber Architecture Guides Tendon-Derived Cells and Adipose Stem Cells into Artificial Tendon Constructs.

Tendon and ligament (T/L) function is intrinsically related with their unique hierarchically and anisotropically organized extracellular matrix. Their natural healing capacity is, however, limited. Here, continuous and aligned electrospun nanofiber threads (CANT) based on synthetic/natural polymer blends mechanically reinforced with cellulose nanocrystals are produced to replicate the nanoscale collagen fibrils grouped into microscale collagen fibers that compose the native T/L. CANT are then incrementally assembled into 3D hierarchical scaffolds, resulting in woven constructions, which simultaneously mimic T/L nano-to-macro architecture, nanotopography, and nonlinear biomechanical behavior. Biological performance is assessed using human-tendon-derived cells (hTDCs) and human adipose stem cells (hASCs). Scaffolds nanotopography and microstructure induce a high cytoskeleton elongation and anisotropic organization typical of tendon tissues. Moreover, the expression of tendon-related markers (Collagen types I and III, Tenascin-C, and Scleraxis) by both cell types, and the similarities observed on their expression patterns over time suggest that the developed scaffolds not only prevent the phenotypic drift of hTDCs, but also trigger tenogenic differentiation of hASCs. Overall, these results demonstrate a feasible approach for the scalable production of 3D hierarchical scaffolds that exhibit key structural and biomechanical properties, which can be advantageously explored in acellular and cellular T/L TE strategies.

Development of Injectable Hyaluronic Acid/Cellulose Nanocrystals Bionanocomposite Hydrogels for Tissue Engineering Applications.

Injectable hyaluronic acid (HA)-based hydrogels compose a promising class of materials for tissue engineering and regenerative medicine applications. However, their limited mechanical properties restrict the potential range of application. In this study, cellulose nanocrystals (CNCs) were employed as nanofillers in a fully biobased strategy for the production of reinforced HA nanocomposite hydrogels. Herein we report the development of a new class of injectable hydrogels composed of adipic acid dihydrazide-modified HA (ADH-HA) and aldehyde-modified HA (a-HA) reinforced with varying contents of aldehyde-modified CNCs (a-CNCs). The obtained hydrogels were characterized in terms of internal morphology, mechanical properties, swelling, and degradation behavior in the presence of hyaluronidase. Our findings suggest that the incorporation of a-CNCs in the hydrogel resulted in a more organized and compact network structure and led to stiffer hydrogels (maximum storage modulus, E', of 152.4 kPa for 0.25 wt % a-CNCs content) with improvements of E' up to 135% in comparison to unfilled hydrogels. In general, increased amounts of a-CNCs led to lower equilibrium swelling ratios and higher resistance to degradation. The biological performance of the developed nanocomposites was assessed toward human adipose derived stem cells (hASCs). HA-CNCs nanocomposite hydrogels exhibited preferential cell supportive properties in in vitro culture conditions due to higher structural integrity and potential interaction of microenvironmental cues with CNC's sulfate groups. hASCs encapsulated in HA-CNCs hydrogels demonstrated the ability to spread within the volume of gels and exhibited pronounced proliferative activity. Together, these results demonstrate that the proposed strategy is a valuable toolbox for fine-tuning the structural, biomechanical, and biochemical properties of injectable HA hydrogels, expanding their potential range of application in the biomedical field.

The potential of cellulose nanocrystals in tissue engineering strategies.

Cellulose nanocrystals (CNCs) are a renewable nanosized raw material that is drawing a tremendous level of attention from the materials community. These rod-shaped nanocrystals that can be produced from a variety of highly available and renewable cellulose-rich sources are endowed with exceptional physicochemical properties which have promoted their intensive exploration as building blocks for the design of a broad range of new materials in the past few decades. However, only recently have these nanosized substrates been considered for bioapplications following the knowledge on their low toxicity and ecotoxicological risk. This Review provides an overview on the recent developments on CNC-based functional biomaterials with potential for tissue engineering (TE) applications, focusing on nanocomposites obtained through different processing technologies usually employed in the fabrication of TE scaffolds into various formats, namely, dense films and membranes, hierarchical three-dimensional (3D) porous constructs (micro/nanofibers mats, foams and sponges), and hydrogels. Finally, while highlighting the major achievements and potential of the reviewed work on cellulose nanocrystals, alternative applications for some of the developed materials are provided, and topics for future research to extend the use of CNCs-based materials in the scope of the TE field are identified.

Human Tendon-on-Chip: Unveiling the Effect of Core Compartment-T Cell Spatiotemporal Crosstalk at the Onset of Tendon Inflammation.

The lack of representative in vitro models recapitulating human tendon (patho)physiology is among the major factors hindering consistent progress in the knowledge-based development of adequate therapies for tendinopathy.Here, an organotypic 3D tendon-on-chip model is designed that allows studying the spatiotemporal dynamics of its cellular and molecular mechanisms.Combining the synergistic effects of a bioactive hydrogel matrix with the biophysical cues of magnetic microfibers directly aligned on the microfluidic chip, it is possible to recreate the anisotropic architecture, cell patterns, and phenotype of tendon intrinsic (core) compartment. When incorporated with vascular-like vessels emulating the interface between its intrinsic-extrinsic compartments, crosstalk with endothelial cells are found to drive stromal tenocytes toward a reparative profile. This platform is further used to study adaptive immune cell responses at the onset of tissue inflammation, focusing on interactions between tendon compartment tenocytes and circulating T cells.The proinflammatory signature resulting from this intra/inter-cellular communication induces the recruitment of T cells into the inflamed core compartment and confirms the involvement of this cellular crosstalk in positive feedback loops leading to the amplification of tendon inflammation.Overall, the developed 3D tendon-on-chip provides a powerful new tool enabling mechanistic studies on the pathogenesis of tendinopathy as well as for assessing new therapies.

Hierarchical Design of Tissue-Mimetic Fibrillar Hydrogel Scaffolds.

Most tissues of the human body present hierarchical fibrillar extracellular matrices (ECMs) that have a strong influence over their physicochemical properties and biological behavior. Of great interest is the introduction of this fibrillar structure to hydrogels, particularly due to the water-rich composition, cytocompatibility, and tunable properties of this class of biomaterials. Here, the main bottom-up fabrication strategies for the design and production of hierarchical biomimetic fibrillar hydrogels and their most representative applications in the fields of tissue engineering and regenerative medicine are reviewed. For example, the controlled assembly/arrangement of peptides, polymeric micelles, cellulose nanoparticles (NPs), and magnetically responsive nanostructures, among others, into fibrillar hydrogels is discussed, as well as their potential use as fibrillar-like hydrogels (e.g., those from cellulose NPs) with key biofunctionalities such as electrical conductivity or remote stimulation. Finally, the major remaining barriers to the clinical translation of fibrillar hydrogels and potential future directions of research in this field are discussed.

A dive into the bath: embedded 3D bioprinting of freeform in vitro models.

Designing functional, vascularized, human scale in vitro models with biomimetic architectures and multiple cell types is a highly promising strategy for both a better understanding of natural tissue/organ development stages to inspire regenerative medicine, and to test novel therapeutics on personalized microphysiological systems. Extrusion-based 3D bioprinting is an effective biofabrication technology to engineer living constructs with predefined geometries and cell patterns. However, bioprinting high-resolution multilayered structures with mechanically weak hydrogel bioinks is challenging. The advent of embedded 3D bioprinting systems in recent years offered new avenues to explore this technology for in vitro modeling. By providing a stable, cell-friendly and perfusable environment to hold the bioink during and after printing, it allows to recapitulate native tissues' architecture and function in a well-controlled manner. Besides enabling freeform bioprinting of constructs with complex spatial organization, support baths can further provide functional housing systems for their long-term in vitro maintenance and screening. This minireview summarizes the recent advances in this field and discuss the enormous potential of embedded 3D bioprinting technologies as alternatives for the automated fabrication of more biomimetic in vitro models.

Writing 3D In Vitro Models of Human Tendon within a Biomimetic Fibrillar Support Platform.

Tendinopathies are poorly understood diseases for which treatment remains challenging. Relevant in vitro models to study human tendon physiology and pathophysiology are therefore highly needed. Here we propose the automated 3D writing of tendon microphysiological systems (MPSs) embedded in a biomimetic fibrillar support platform based on cellulose nanocrystals (CNCs) self-assembly. Tendon decellularized extracellular matrix (dECM) was used to formulate bioinks that closely recapitulate the biochemical signature of tendon niche. A monoculture system recreating the cellular patterns and phenotype of the tendon core was first developed and characterized. This system was then incorporated with a vascular compartment to study the crosstalk between the two cell populations. The combined biophysical and biochemical cues of the printed pattern and dECM hydrogel were revealed to be effective in inducing human-adipose-derived stem cells (hASCs) differentiation toward the tenogenic lineage. In the multicellular system, chemotactic effects promoted endothelial cells migration toward the direction of the tendon core compartment, while the established cellular crosstalk boosted hASCs tenogenesis, emulating the tendon development stages. Overall, the proposed concept is a promising strategy for the automated fabrication of humanized organotypic tendon-on-chip models that will be a valuable new tool for the study of tendon physiology and pathogenesis mechanisms and for testing new tendinopathy treatments.

Supercritical fluid extraction of Eucalyptus globulus bark-A promising approach for triterpenoid production.

Eucalyptus bark contains significant amounts of triterpenoids with demonstrated bioactivity, namely triterpenic acids and their acetyl derivatives (ursolic, betulinic, oleanolic, betulonic, 3-acetylursolic, and 3-acetyloleanolic acids). In this work, the supercritical fluid extraction (SFE) of Eucalyptus globulus deciduous bark was carried out with pure and modified carbon dioxide to recover this fraction, and the results were compared with those obtained by Soxhlet extraction with dichloromethane. The effects of pressure (100-200 bar), co-solvent (ethanol) content (0, 5 and 8% wt), and multistep operation were studied in order to evaluate the applicability of SFE for their selective and efficient production. The individual extraction curves of the main families of compounds were measured, and the extracts analyzed by GC-MS. Results pointed out the influence of pressure and the important role played by the co-solvent. Ethanol can be used with advantage, since its effect is more important than increasing pressure by several tens of bar. At 160 bar and 40 °C, the introduction of 8% (wt) of ethanol greatly improves the yield of triterpenoids more than threefold.

The tendon microenvironment: Engineered in vitro models to study cellular crosstalk.

Tendinopathy is a multi-faceted pathology characterized by alterations in tendon microstructure, cellularity and collagen composition. Challenged by the possibility of regenerating pathological or ruptured tendons, the healing mechanisms of this tissue have been widely researched over the past decades. However, so far, most of the cellular players and processes influencing tendon repair remain unknown, which emphasizes the need for developing relevant in vitro models enabling to study the complex multicellular crosstalk occurring in tendon microenvironments. In this review, we critically discuss the insights on the interaction between tenocytes and the other tendon resident cells that have been devised through different types of existing in vitro models. Building on the generated knowledge, we stress the need for advanced models able to mimic the hierarchical architecture, cellularity and physiological signaling of tendon niche under dynamic culture conditions, along with the recreation of the integrated gradients of its tissue interfaces. In a forward-looking vision of the field, we discuss how the convergence of multiple bioengineering technologies can be leveraged as potential platforms to develop the next generation of relevant in vitro models that can contribute for a deeper fundamental knowledge to develop more effective treatments.

Texturing Hierarchical Tissues by Gradient Assembling of Microengineered Platelet-Lysates Activated Fibers.

The heterogeneity of hierarchical tissues requires designing multipart engineered constructs as suitable tissue replacements. Herein, the incorporation of platelet lysate (PL) within an electrospun fiber core is proposed aiming for the fabrication of functionally graded 3D scaffolds for heterotypic tissues regeneration, such as tendon-to-bone interfaces. First, anisotropic yarns (A-Yarns) and isotropic threads with nanohydroxyapatite (I-Threads/PL@nHAp) are fabricated to recreate the tendon- and bone-microstructures and both incorporated with PL using emulsion electrospinning for a sustained and local delivery of growth factors, cytokines, and chemokines. Biological performance using human adipose-derived stem cells demonstrates that A-Yarns/PL induce a higher expression of scleraxis, a tenogenic-marker, while in I-Threads/PL@nHAp, higher alkaline phosphatase activity and matrix mineralization suggest an osteogenic commitment without the need for biochemical supplementation compared to controls. As a proof-of-concept, functional 3D gradient scaffolds are fabricated using a weaving technique, resulting in 3D textured hierarchical constructs with gradients in composition and topography. Additionally, the precise delivery of bioactive cues together with in situ biophysical features guide the commitment into a phenotypic gradient exhibiting chondrogenic and osteochondrogenic profiles in the interface of scaffolds. Overall, a promising patch solution for the regeneration of tendon-to-bone tissue interface through the fabrication of PL-functional 3D gradient constructs is demonstrated.

Bioengineered 3D Living Fibers as In Vitro Human Tissue Models of Tendon Physiology and Pathology.

Clinically relevant in vitro models of human tissue's health and disease are urgently needed for a better understanding of biological mechanisms essential for the development of novel therapies. Herein, physiological (healthy) and pathological (disease) tendon states are bioengineered by coupling the biological signaling of platelet lysate components with controlled 3D architectures of electrospun microfibers to drive the fate of human tendon cells in different composite living fibers (CLFs). In the CLFs-healthy model, tendon cells adopt a high cytoskeleton alignment and elongation, express tendon-related markers (scleraxis, tenomodulin, and mohawk) and deposit a dense tenogenic matrix. In contrast, cell crowding with low preferential orientation, high matrix deposition, and phenotypic drift leading to increased expression of nontendon related and fibrotic markers, are characteristics of the CLFs-diseased model. This diseased-like profile, also reflected in the increase of COL3/COL1 ratio, is further evident by the imbalance between matrix remodeling and degradation effectors, characteristic of tendinopathy. In summary, microengineered 3D in vitro models of human tendon healthy and diseased states are successfully fabricated. Most importantly, these innovative and versatile microphysiological models offer major advantages over currently used systems, holding promise for drugs screening and development of new therapies.

Controlling the fate of regenerative cells with engineered platelet-derived extracellular vesicles.

Extracellular vesicles (EVs) have emerged as cell-free nanotherapeutic agents for the potential treatment of multiple diseases and for tissue engineering and regenerative medicine strategies. Nevertheless, the field has typically relied on EVs derived from stem cells, the production of which in high quantities and high reproducibility is still under debate. Platelet-derived EVs were produced by a freeze-thaw method of platelet concentrates, a highly available clinical waste material. The aim of this study was to produce and thoroughly characterize platelet-derived EVs and understand their effects in adipose-tissue derived stem cells (hASCs), endothelial cells (HUVECs) and macrophages. Two different EV populations were obtained after differential centrifugation, namely small EVs (sEVs) and medium EVs (mEVs), which showed different size distributions and unique proteomic signatures. EV interaction with hASCs resulted in the modulation of the gene expression of markers related to their commitment toward different lineages. Moreover, mEVs showed higher angiogenic potential than sEVs, in a tube formation assay with HUVECs. Also, the EVs were able to modulate macrophage polarization. Altogether, these results suggest that platelet-derived EVs are promising candidates to be used as biochemical signals or therapeutic tools in tissue engineering and regenerative medicine approaches.

Xeno-free bioengineered human skeletal muscle tissue using human platelet lysate-based hydrogels.

Bioengineered human skeletal muscle tissues have emerged in the last years as newin vitrosystems for disease modeling. These bioartificial muscles are classically fabricated by encapsulating human myogenic precursor cells in a hydrogel scaffold that resembles the extracellular matrix. However, most of these hydrogels are derived from xenogenic sources, and the culture media is supplemented with animal serum, which could interfere in drug testing assays. On the contrary, xeno-free biomaterials and culture conditions in tissue engineering offer increased relevance for developing human disease models. In this work, we used human platelet lysate (PL)-based nanocomposite hydrogels (HUgel) as scaffolds for human skeletal muscle tissue engineering. These hydrogels consist of human PL reinforced with aldehyde-cellulose nanocrystals (a-CNC) that allow tunable mechanical, structural, and biochemical properties for the 3D culture of stem cells. Here, we developed hydrogel casting platforms to encapsulate human muscle satellite stem cells in HUgel. The a-CNC content was modulated to enhance matrix remodeling, uniaxial tension, and self-organization of the cells, resulting in the formation of highly aligned, long myotubes expressing sarcomeric proteins. Moreover, the bioengineered human muscles were subjected to electrical stimulation, and the exerted contractile forces were measured in a non-invasive manner. Overall, our results demonstrated that the bioengineered human skeletal muscles could be built in xeno-free cell culture platforms to assess tissue functionality, which is promising for drug development applications.

Highly elastic and bioactive bone biomimetic scaffolds based on platelet lysate and biomineralized cellulose nanocrystals.

Bone is a vascularized organic-inorganic composite tissue that shows a heavily-mineralized extracellular matrix (ECM) on the nanoscale. Herein, the nucleation of calcium phosphates during the biomineralization process was mimicked using negatively-charged cellulose nanocrystals (CNCs). These mineralized-CNCs were combined with platelet lysate to produce nanocomposite scaffolds through cryogelation to mimic bone ECM protein-mineral composite nature and take advantage of the bioactivity steaming from platelet-derived biomolecules. The nanocomposite scaffolds showed high microporosity (94-95%), high elasticity (recover from 75% strain cycles), injectability, and modulated platelet-derived growth factors sequestration and release. Furthermore, they increased alkaline phosphatase activity (up to 10-fold) and up-regulated the expression of bone-related markers (up to 2-fold), without osteogenic supplementation, demonstrating their osteoinductive properties. Also, the scaffolds promoted the chemotaxis of endothelial cells and enhanced the expression of endothelial markers, showing proangiogenic potential. These results suggest that the mineralized nanocomposite scaffolds can enhance bone regeneration by simultaneously promoting osteogenesis and angiogenesis.