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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., (© 2024 The Author(s). Advanced Science published by Wiley‐VCH GmbH.)

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., (© 2024 The Authors. Advanced Healthcare Materials published by Wiley‐VCH GmbH.)

The paradigm shift in the endodontic field from replacement toward regenerative therapies has witnessed the ever-growing research in tissue engineering and regenerative medicine targeting pulp-dentin complex in the past few years. Abundant literature on the subject that has been produced, however, is scattered over diverse areas of knowledge. Moreover, the terminology and concepts are not always consensual, reflecting the range of research fields addressing this subject, from endodontics to biology, genetics, and engineering, among others. This fact triggered some misinterpretations, mainly when the denominations of different approaches were used as synonyms. The evaluation of results is not precise, leading to biased conjectures. Therefore, this literature review aims to conceptualize the commonly used terminology, summarize the main research areas on pulp regeneration, identify future trends, and ultimately clarify whether we are really on the edge of a paradigm shift in contemporary endodontics toward pulp regeneration.

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.

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.

Tendons are mechanosensitive connective tissues responsible for the connection between muscles and bones by transmitting forces that allow the movement of the body, yet, with advancing age, tendons become more prone to degeneration followed by injuries. Tendon diseases are one of the main causes of incapacity worldwide, leading to changes in tendon composition, structure, and biomechanical properties, as well as a decline in regenerative potential. There is still a great lack of knowledge regarding tendon cellular and molecular biology, interplay between biochemistry and biomechanics, and the complex pathomechanisms involved in tendon diseases. Consequently, this reflects a huge need for basic and clinical research to better elucidate the nature of healthy tendon tissue and also tendon aging process and associated diseases. This chapter concisely describes the effects that the aging process has on tendons at the tissue, cellular, and molecular levels and briefly reviews potential biological predictors of tendon aging. Recent research findings that are herein reviewed and discussed might contribute to the development of precision tendon therapies targeting the elderly population., (© 2023. The Author(s), under exclusive license to Springer Nature Switzerland AG.)

Dental implants, usually made of titanium, are exposed to hostile oral microflora that facilitate bacterial infections and subsequent inflammation. To mitigate these processes, we coated titanium substrates with block copolymer nanopatterns and investigated the bactericidal effect of these coatings against Gram-positive and Gram-negative bacteria. We found that the bactericidal efficacy of the coatings depends on their morphology and surface chemistry as well as on the bacterial strain: an optimal combination can lead to significant bacterial death for a short time, i.e. 90% for 90 min. Human gingival fibroblasts in contact with the nanopatterned coatings showed similar cell attachment and morphology as on bare Ti. Immunostaining assays showed similar levels of CCR7 and CD206 in macrophages cultured over the nanopatterns and bare Ti, demonstrating adequate properties for tissue integration. The nanopatterns induced a small increase in macrophage aspect ratio, which might indicate early states of M2 polarization, given the absence of CD206.

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., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

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., (© 2022 Wiley-VCH GmbH.)

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., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Metallic implants are widely used in diverse clinical applications to aid in recovery from lesions or to replace native hard tissues. However, the lack of integration of metallic surfaces with soft tissue interfaces causes the occurrence of biomaterial-associated infections, which can trigger a complicated inflammatory response and, ultimately, implant failure. Here, a multifunctional implant surface showing nanoscale anisotropy, based on the controlled deposition of cellulose nanocrystals (CNC), and biological activity derived from platelet lysate (PL) biomolecules sequestered and presented on CNC surface, is proposed. The anisotropic radial nanopatterns are produced on polished titanium surfaces by spin-coating CNC at high speed. Furthermore, CNC surface chemistry allows to further sequester and form a coating of bioactive molecules derived from PL. The surface anisotropy provided by CNC guides fibroblasts growth and alignment up to 14 days of culture. Moreover, PL-derived biomolecules polarize macrophages toward the M2-like anti-inflammatory phenotype. These results suggest that the developed multifunctional surfaces can promote soft tissue integration to metallic implants and, at the same time, prevent bacterial invasion, tissue inflammation, and failure of biomedical metallic implants., (© 2021 Wiley-VCH GmbH.)

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.

In the quest for finding new strategies to enhance tissue integration and to reduce the risk of bacterial colonization around endosseous implants, we report the application of auto-oxidative phenolic coatings made of tannic acid and pyrogallol to titanium surfaces. The functionalized surfaces were screened for their biological performance using cultures of primary human osteoblasts and biofilm-forming bioluminescent staphylococci S. epidermidis Xen43 and S. aureus Xen29. No toxic effect of the coatings on osteoblasts was detected. While tannic acid coatings seemed to induce a delay in osteoblast maturation, they revealed anti-inflammatory potential. Similar effects were observed for pyrogallol coatings deposited for 24 h. Thin pyrogallol coatings deposited for 2 h seemed to promote osteoblast maturation and revealed increased calcium deposition. The effects on osteoblast were found to be related to the release of phenolic compounds from the surfaces. While the phenolic coatings could not inhibit staphylococcal biofilm formation on the titanium surfaces, released phenolic compounds had an inhibitory effect the growth of planktonic bacteria. In conclusion, the assessed coating systems represent a versatile functionalization method which exhibit promising effects for endosseous implant applications.

Injectable hydrogels are particularly interesting for applications in minimally invasive tissue engineering and regenerative medicine strategies. However, the typical isotropic microstructure of these biomaterials limits their potential for the regeneration of ordered tissues. In the present work, we decorated rod-shaped cellulose nanocrystals with magnetic nanoparticles and coated these with polydopamine and polyethylene glycol polymer brushes to obtain chemical and colloidal stable nanoparticles. Then, these nanoparticles (0.1-0.5 wt %) were incorporated within gelatin hydrogels, creating injectable and magnetically responsive materials with potential for various biomedical applications. Nanoparticle alignment within the hydrogel matrix was achieved under exposure to uniform low magnetic fields (108 mT), resulting in biomaterials with directional microstructure and anisotropic mechanical properties. The biological performance of these nanocomposite hydrogels was studied using adipose tissue derived human stem cells. Cells encapsulated in the nanocomposite hydrogels showed high rates of viability demonstrating that the nanocomposite biomaterials are not cytotoxic. Remarkably, the microstructural patterns stemming from nanoparticle alignment induced the directional growth of seeded and, to a lower extent, encapsulated cells in the hydrogels, suggesting that this injectable system might find application in both cellular and acellular strategies targeting the regeneration of anisotropic tissues.

Clinical summary: A tissue engineering approach was used to aid the surgical repair of a chronic oronasal fistula (ONF) in a 13-year-old cat. A three-dimensional (3D) printed mesh, tailored to the size and shape of the ONF, was created to support a soft tissue flap used to close the defect; and also to provide a matrix for mesenchymal stromal cells present in bone marrow aspirate and bioactive cytokines and growth factors present in platelet-rich fibrin harvested from the patient. A CT scan at day 75 after surgery revealed the formation of new tissue in the defect and the healing process was complete at follow-up 6 months after surgery. Relevance and novel information: Complications are frequently reported following surgical repair of ONFs and include dehiscence of the palatal suture line, flap necrosis due to damage to the greater palatine artery and maxillary osteomyelitis, mainly due to chronic infection and bone lysis. The case described here demonstrates how input from a multidisciplinary team and the use of a biomaterial, processed by sophisticated technologies, can create a precision regenerative medicine strategy adapted to the patient's clinical needs; this provided a novel therapeutic solution for an otherwise hard to treat clinical problem.

Many dental implants fail due to the infection and inflammation that walk hand in hand with poor healing and soft tissue integration. Titanium surfaces were nanocoated with quercitrin, a natural flavonoid, with the aim to improve soft tissue integration and increase dental implants success. Streptococcus mutans attachment and biofilm formation was analysed. Then, the anti-inflammatory properties and the potential of quercitrin-nanocoated surfaces to boost soft tissue regeneration were tested using human gingival fibroblasts. An inflammatory situation was mimicked using interleulin-1-beta. We found that quercitrin-nanocoated surfaces decreased initial bacterial adhesion while increasing human gingival fibroblasts attachment. Furthermore, quercitrin-nanocoated Ti increased collagen mRNA levels and decreased matrix metalloproteinase-1/tissue inhibitor of metalloproteinanse-1 mRNA ratio, which is related to a reduced metalloproteinase-mediated collagen degradation, while also decreasing the pro-inflammatory prostaglandin E2 release under basal and inflammatory conditions. These results suggest that quercitrin-nanocoated surfaces could enhance the soft tissue integration and increase dental implants success.

Purpose: Previous studies have demonstrated the capacity of a designed proline-rich synthetic peptide to stimulate osteoblast differentiation and biomineralization in vitro. Therefore, the aim of the present study was to evaluate the osseointegration capacity of titanium (Ti) implants coated with these peptides in a rabbit model., Materials and Methods: Four calibrated defects were prepared in the tibiae of three New Zealand rabbits, and the defects were randomized into a test group (peptide-modified machined Ti implant) and a control group (unmodified machined Ti implant). The performance in vivo was investigated after 4 weeks of implantation by real-time reverse transcriptase polymerase chain reaction of bone and inflammatory markers, microcomputed tomographic analysis of mineralized bone, and histologic examination., Results: The peptides adsorbed in agglomerates on Ti and underwent a change in secondary structure upon adsorption, which induced an increase in surface wettability. Gene expression markers indicated that peptide-coated Ti implants had significantly decreased mRNA levels of tartrate-resistant acid phosphatase. A trend toward increased osteocalcin in the peri-implant bone tissue was also seen. Bone morphometric and histologic parameters did not show significant differences, although the peptide group showed a higher percentage of new bone histologically., Conclusions: Proline-rich peptides have potential as a biocompatible coating for promoting osseointegration of Ti implants by reducing bone resorption.

2-Hydroxyoleic acid (2OHOA) is a synthetic fatty acid with antihypertensive properties that is able to alter structural membranes properties. The main purpose of this study was to analyze the effect of 2OHOA on the membrane architecture in cholesterol (Cho)-rich domains. For this purpose, model membranes mimicking the composition of lipid rafts and PC- or PE-Cho-rich domains were examined in the absence and presence of 2OHOA by synchrotron X-ray diffraction, atomic force microscopy (AFM) and microcalorimetry (DSC) techniques. Our results demonstrate that 2OHOA phase separates from lipid raft domains and affects the lateral organization of lipids in the membrane. In model raft membranes, 2OHOA interacted with the sphingomyelin (SM) gel phase increasing the thickness of the water layer, which should lead to increased bilayer fluidity. The hydrogen binding competition between 2OHOA and Cho could favour the enrichment of 2OHOA in SM domains separated from the SM-Cho domains, resulting in an enhanced phase separation into SM-2OHOA-rich liquid-disordered (non-raft) and SM-Cho-rich liquid-ordered (raft) domains. The segregation into 2OHOA-rich/Cho-poor and 2OHOA-poor/Cho-rich domains was also observed in PC bilayers.

Colonization of implant surfaces with bacteria should ideally be prevented right from implantation, as bacteria attaching to the surface will form a biofilm, being then well protected against antibiotic treatment. Therefore, implant coatings should combine antibacterial properties with biocompatibility towards their host tissue. We tested a UV-induced covalent coating procedure with eicosapentaenoic acid (EPA) for smooth titanium (Ti) surfaces for its ability to prevent attachment and proliferation of Staphylococcus epidermidis and to allow mineralization of MC3T3-E1 osteoblasts. Bacterial initial attachment was highest for EPA-coated surfaces, but was reduced by vigorous washing, possibly due to low adhesive strength on those surfaces. We found an increase in the ratio of dead bacteria and in overall biofilm after 16 h on Ti surfaces with covalently bound EPA compared to Ti. The UV-induced EPA coating did not impair the ability of MC3T3-E1 preosteoblasts to mineralize, while a reduction in mineralization could be found for UV-irradiated Ti surfaces and UV-irradiated surfaces washed with ethanol compared to Ti. Although in vivo studies are needed to evaluate the clinical significance, our results indicate that covalent coating of Ti surfaces with EPA by UV irradiation decreases the survival of S. epidermidis and maintains the mineralization ability of osteoblasts., (Copyright © 2012 Orthopaedic Research Society.)

Free triterpenic acids (TTPs) present in plants are bioactive compounds exhibiting multiple nutriceutical activities. The underlying molecular mechanisms have only been examined in part and mainly focused on anti-inflammatory properties, cancer and cardiovascular diseases, in all of which TTPs frequently affect membrane-related proteins. Based on the structural characteristics of TTPs, we assume that their effect on biophysical properties of cell membranes could play a role for their biological activity. In this context, our study is focused on the compounds, oleanolic (3β-hydroxy-12-oleanen-28-oic acid, OLA), maslinic (2α,3β-dihydroxy-12-oleanen-28-oic acid, MSL) and ursolic ((3β)-3-hydroxyurs-12-en-28-oic acid, URL) as the most important TTPs present in orujo olive oil. X-ray diffraction, differential scanning calorimetry, (31)P nuclear magnetic resonance and Laurdan fluorescence data provide experimental evidence that OLA, MSL and URL altered the structural properties of 1,2-dipalmitoyl-sn-glycero-3-phosphatidylcholine (DPPC) and DPPC-Cholesterol (Cho) rich membranes, being located into the polar-hydrophobic interphase. Specifically, in DPPC membranes, TTPs altered the structural order of the L(β'), phase without destabilizing the lipid bilayer. The existence of a nonbilayer isotropic phase in coexistence with the liquid crystalline L(α) phase, as observed in DPPC:URL samples, indicated the presence of lipid structures with high curvature (probably inverted micelles). In DPPC:Cho membranes, TTPs affected the membrane phase properties increasing the Laurdan GP values above 40°C. MSL and URL induced segregation of Cho within the bilayer, in contrast to OLA, that reduced the structural organization of the membrane. These results strengthen the relevance of TTP interactions with cell membranes as a molecular mechanism underlying their broad spectrum of biological effects., (Copyright © 2010 Elsevier B.V. All rights reserved.)