Bioengineered niches that recreate physiological extracellular matrix organisation to support long-term haematopoietic stem cells.

Long-term reconstituting haematopoietic stem cells (LT-HSCs) are used to treat blood disorders via stem cell transplantation. The very low abundance of LT-HSCs and their rapid differentiation during in vitro culture hinders their clinical utility. Previous developments using stromal feeder layers, defined media cocktails, and bioengineering have enabled HSC expansion in culture, but of mostly short-term HSCs and progenitor populations at the expense of naive LT-HSCs. Here, we report the creation of a bioengineered LT-HSC maintenance niche that recreates physiological extracellular matrix organisation, using soft collagen type-I hydrogels to drive nestin expression in perivascular stromal cells (PerSCs). We demonstrate that nestin, which is expressed by HSC-supportive bone marrow stromal cells, is cytoprotective and, via regulation of metabolism, is important for HIF-1α expression in PerSCs. When CD34+ve HSCs were added to the bioengineered niches comprising nestin/HIF-1α expressing PerSCs, LT-HSC numbers were maintained with normal clonal and in vivo reconstitution potential, without media supplementation. We provide proof-of-concept that our bioengineered niches can support the survival of CRISPR edited HSCs. Successful editing of LT-HSCs ex vivo can have potential impact on the treatment of blood disorders.

Developing and Investigating a Nanovibration Intervention for the Prevention/Reversal of Bone Loss Following Spinal Cord Injury.

Osteoporosis disrupts the fine-tuned balance between bone formation and resorption, leading to reductions in bone quantity and quality and ultimately increasing fracture risk. Prevention and treatment of osteoporotic fractures is essential for reductions in mortality, morbidity, and the economic burden, particularly considering the aging global population. Extreme bone loss that mimics time-accelerated osteoporosis develops in the paralyzed limbs following complete spinal cord injury (SCI). In vitro nanoscale vibration (1 kHz, 30 or 90 nm amplitude) has been shown to drive differentiation of mesenchymal stem cells toward osteoblast-like phenotypes, enhancing osteogenesis and inhibiting osteoclastogenesis simultaneously. Here, we develop and characterize a wearable device designed to deliver and monitor continuous nanoamplitude vibration to the hindlimb long bones of rats with complete SCI. We investigate whether a clinically feasible dose of nanovibration (two 2 h/day, 5 days/week for 6 weeks) is effective at reversing the established SCI-induced osteoporosis. Laser interferometry and finite element analysis confirmed transmission of nanovibration into the bone, and microcomputed tomography and serum bone formation and resorption markers assessed effectiveness. The intervention did not reverse SCI-induced osteoporosis. However, serum analysis indicated an elevated concentration of the bone formation marker procollagen type 1 N-terminal propeptide (P1NP) in rats receiving 40 nm amplitude nanovibration, suggesting increased synthesis of type 1 collagen, the major organic component of bone. Therefore, enhanced doses of nanovibrational stimulus may yet prove beneficial in attenuating/reversing osteoporosis, particularly in less severe forms of osteoporosis.

Hybrid Micro-/Nanoprotein Platform Provides Endocrine-like and Extracellular Matrix-like Cell Delivery of Growth Factors.

Protein materials are versatile tools in diverse biomedical fields. Among them, artificial secretory granules (SGs), mimicking those from the endocrine system, act as mechanically stable reservoirs for the sustained release of proteins as oligomeric functional nanoparticles. Only validated in oncology, the physicochemical properties of SGs, along with their combined drug-releasing and scaffolding abilities, make them suitable as smart topographies in regenerative medicine for the prolonged delivery of growth factors (GFs). Thus, considering the need for novel, safe, and cost-effective materials to present GFs, in this study, we aimed to biofabricate a protein platform combining both endocrine-like and extracellular matrix fibronectin-derived (ECM-FN) systems. This approach is based on the sustained delivery of a nanostructured histidine-tagged version of human fibroblast growth factor 2. The GF is presented onto polymeric surfaces, interacting with FN to spontaneously generate nanonetworks that absorb and present the GF in the solid state, to modulate mesenchymal stromal cell (MSC) behavior. The results show that SGs-based topographies trigger high rates of MSCs proliferation while preventing differentiation. While this could be useful in cell therapy manufacture demanding large numbers of unspecialized MSCs, it fully validates the hybrid platform as a convenient setup for the design of biologically active hybrid surfaces and in tissue engineering for the controlled manipulation of mammalian cell growth.

Building bones for blood and beyond: the growing field of bone marrow niche model development.

The bone marrow (BM) niche is a complex microenvironment that provides the signals required for regulation of hematopoietic stem cells (HSCs) and the process of hematopoiesis they are responsible for. Bioengineered models of the BM niche incorporate various elements of the in vivo BM microenvironment, including cellular components, soluble factors, a three-dimensional environment, mechanical stimulation of included cells, and perfusion. Recent advances in the bioengineering field have resulted in a spate of new models that shed light on BM function and are approaching precise imitation of the BM niche. These models promise to improve our understanding of the in vivo microenvironment in health and disease. They also aim to serve as platforms for HSC manipulation or as preclinical models for screening novel therapies for BM-associated disorders and diseases.

Gallium and silver-doped titanium surfaces provide enhanced osteogenesis, reduce bone resorption and prevent bacterial infection in co-culture.

Bacterial infection remains a significant problem associated with orthopaedic surgeries leading to surgical site infection (SSI). This unmet medical need can become an even greater complication when surgery is due to malignant bone tumor. In the present study, we evaluated in vitro titanium (Ti) implants subjected to gallium (Ga) and silver (Ag)-doped thermochemical treatment as strategy to prevent SSI and improve osteointegration in bone defects caused by diseases such as osteoporosis, bone tumor, or bone metastasis. Firstly, as Ga has been reported to be an osteoinductive and anti-resorptive agent, its performance in the mixture was proved by studying human mesenchymal stem cells (hMSC) and pre-osteoclasts (RAW264.7) behaviour. Then, the antibacterial potential provided by Ag was assessed by resembling "The Race for the Surface" between hMSC and Pseudomonas aeruginosa in two co-culture methods. Moreover, the presence of quorum sensing molecules in the co-culture was evaluated. The results highlighted the suitability of the mixture to induce osteodifferentiation and reduce osteoclastogenesis in vitro. Furthermore, the GaAg surface promoted strong survival rate and retained osteoinduction potential of hMSCs even after bacterial inoculation. Therefore, GaAg-modified titanium may be an ideal candidate to repair bone defects caused by excessive bone resorption, in addition to preventing SSI. STATEMENT OF SIGNIFICANCE: This article provides important insights into titanium for fractures caused by osteoporosis or bone metastases with high incidence in surgical site infection (SSI) because in this situation bacterial infection can become a major disaster. In order to solve this unmet medical need, we propose a titanium implant modified with gallium and silver to improve osteointegration, reduce bone resorption and avoid bacterial infection. For that aim, we study osteoblast and osteoclast behavior with the main novelty focused on the antibacterial evaluation. In this work, we recreate "the race for the surface" in long-term experiments and study bacterial virulence factors (quorum sensing). Therefore, we believe that our article could be of great interest, providing a great impact on future orthopedic applications.

Mechanotransducive surfaces for enhanced cell osteogenesis, a review.

Novel strategies employing mechano-transducing materials eliciting biological outcomes have recently emerged for controlling cellular behaviour. Targeted cellular responses are achieved by manipulating physical, chemical, or biochemical modification of material properties. Advances in techniques such as nanopatterning, chemical modification, biochemical molecule embedding, force-tuneable materials, and artificial extracellular matrices are helping understand cellular mechanotransduction. Collectively, these strategies manipulate cellular sensing and regulate signalling cascades including focal adhesions, YAP-TAZ transcription factors, and multiple osteogenic pathways. In this minireview, we are providing a summary of the influence that these materials, particularly titanium-based orthopaedic materials, have on cells. We also highlight recent complementary methodological developments including, but not limited to, the use of metabolomics for identification of active biomolecules that drive cellular differentiation.

Strengths and opportunities in research into extracellular matrix ageing: A consultation with the ECMage research community.

Ageing causes progressive decline in metabolic, behavioural, and physiological functions, leading to a reduced health span. The extracellular matrix (ECM) is the three-dimensional network of macromolecules that provides our tissues with structure and biomechanical resilience. Imbalance between damage and repair/regeneration causes the ECM to undergo structural deterioration with age, contributing to age-associated pathology. The ECM 'Ageing Across the Life Course' interdisciplinary research network (ECMage) was established to bring together researchers in the United Kingdom, and internationally, working on the emerging field of ECM ageing. Here we report on a consultation at a joint meeting of ECMage and the Medical Research Council / Versus Arthritis Centre for Integrated Research into Musculoskeletal Ageing, held in January 2023, in which delegates analysed the key questions and research opportunities in the field of ECM ageing. We examine fundamental biological questions, enabling technologies, systems of study and emerging in vitro and in silico models, alongside consideration of the broader challenges facing the field.

Engineered Surfaces That Promote Capture of Latent Proteins to Facilitate Integrin-Mediated Mechanical Activation of Growth Factors.

Conventional osteogenic platforms utilize active growth factors to repair bone defects that are extensive in size, but they can adversely affect patient health. Here, an unconventional osteogenic platform is reported that functions by promoting capture of inactive osteogenic growth factor molecules to the site of cell growth for subsequent integrin-mediated activation, using a recombinant fragment of latent transforming growth factor beta-binding protein-1 (rLTBP1). It is shown that rLTBP1 binds to the growth-factor- and integrin-binding domains of fibronectin on poly(ethyl acrylate) surfaces, which immobilizes rLTBP1 and promotes the binding of latency associated peptide (LAP), within which inactive transforming growth factor beta 1 (TGF-ß1) is bound. rLTBP1 facilitates the interaction of LAP with integrin ß1 and the subsequent mechanically driven release of TGF-ß1 to stimulate canonical TGF-ß1 signaling, activating osteogenic marker expression in vitro and complete regeneration of a critical-sized bone defect in vivo.

Nanotextured titanium inhibits bacterial activity and supports cell growth on 2D and 3D substrate: A co-culture study.

Medical implant-associated infections pose a significant challenge to modern medicine, with aseptic loosening and bacterial infiltration being the primary causes of implant failure. While nanostructured surfaces have demonstrated promising antibacterial properties, the translation of their efficacy from 2D to 3D substrates remains a challenge. Here, we used scalable alkaline etching to fabricate nanospike and nanonetwork topologies on 2D and laser powder-bed fusion printed 3D titanium. The fabricated surfaces were compared with regard to their antibacterial properties against Staphylococcus aureus, Escherichia coli, and Pseudomonas aeruginosa, and mesenchymal stromal cell responses with and without the presence of bacteria. Finite elemental analysis assessed the mechanical properties and permeability of the 3D substrate. Our findings suggest that 3D nanostructured surfaces have potential to both prevent implant infections and allow host cell integration. This work represents a significant step towards developing effective and scalable fabrication methods on 3D substrates with consistent and reproducible antibacterial activity, with important implications for the future of medical implant technology.

Overcoming BCR::ABL1 dependent and independent survival mechanisms in chronic myeloid leukaemia using a multi-kinase targeting approach.

BACKGROUND: Despite improved patient outcome using tyrosine kinase inhibitors (TKIs), chronic myeloid leukaemia (CML) patients require life-long treatment due to leukaemic stem cell (LSC) persistence. LSCs reside in the bone marrow (BM) niche, which they modify to their advantage. The BM provides oncogene-independent signals to aid LSC cell survival and quiescence. The bone-morphogenetic pathway (BMP) is one pathway identified to be highly deregulated in CML, with high levels of BMP ligands detected in the BM, accompanied by CML stem and progenitor cells overexpressing BMP type 1 receptors- activin-like kinases (ALKs), especially in TKI resistant patients. Saracatinib (SC), a SRC/ABL1 dual inhibitor, inhibits the growth of CML cells resistant to the TKI imatinib (IM). Recent studies indicate that SC is also a potent ALK inhibitor and BMP antagonist. Here we investigate the efficacy of SC in overcoming CML BCR::ABL1 dependent and independent signals mediated by the BM niche both in 2D and 3D culture. METHODS: CML cells (K562 cell line and CML CD34+ primary cells) were treated with single or combination treatments of: IM, SC and the BMP receptors inhibitor dorsomorphin (DOR), with or without BMP4 stimulation in 2D (suspension) and 3D co-culture on HS5 stroma cell line and mesenchymal stem cells in AggreWell and microfluidic devices. Flow cytometry was performed to investigate apoptosis, cell cycle progression and proliferation, alongside colony assays following treatment. Proteins changes were validated by immunoblotting and transcriptional changes by Fluidigm multiplex qPCR. RESULTS: By targeting the BMP pathway, using specific inhibitors against ALKs in combination with SRC and ABL TKIs, we show an increase in apoptosis, altered cell cycle regulation, fewer cell divisions, and reduced numbers of CD34+ cells. Impairment of long-term proliferation and differentiation potential after combinatorial treatment also occurred. CONCLUSION: BMP signalling pathway is important for CML cell survival. Targeting SRC, ABL and ALK kinases is more effective than ABL inhibition alone, the combination efficacy importantly being demonstrated in both 2D and 3D cell cultures highlighting the need for combinatorial therapies in contrast to standard of care single agents. Our study provides justification to target multiple kinases in CML to combat LSC persistence.

Blood is made in the spongy inner most section of the bone, called the bone marrow. The bone marrow is where normal blood stem cells live that are responsible for producing the different blood cell types; white blood cells (fight infections), red blood cells (carrying oxygen around the body), platelets (blood clotting) and other cells which support this process. Chronic myeloid leukaemia (CML) is a type of blood cancer that starts in the bone marrow. CML occurs when a normal blood stem cell becomes damaged, forming a leukaemia stem cell (LSC), leading to blood cancer. LSCs multiply and generate many faulty cancerous white blood cells that do not work properly. Patients are treated with a drug called imatinib, which reduces the number of cancerous cells circulating in the body. In many cases, this treatment is not enough to cure the disease because the bone marrow protects the LSCs from the drug meaning patients must remain on long term treatment. This work has discovered one of the ways in which the bone marrow protects LSCs from treatments and has used this knowledge to test new drugs that stop this protection. Our findings show that by combining two drugs, one that overcomes this protection and one that directly targets the cancerous cells, we can destroy more of the LSCs. These findings are a step closer towards a cure for CML and could improve treatment for patients in the future. Video Abstract.

Surface-Modified Piezoelectric Copolymer Poly(vinylidene fluoride-trifluoroethylene) Supporting Physiological Extracellular Matrixes to Enhance Mesenchymal Stem Cell Adhesion for Nanoscale Mechanical Stimulation.

There is an unmet clinical need to provide viable bone grafts for clinical use. Autologous bone, one of the most commonly transplanted tissues, is often used but is associated with donor site morbidity. Tissue engineering strategies to differentiate an autologous cell source, such as mesenchymal stromal cells (MSCs), into a potential bone-graft material could help to fulfill clinical demand. However, osteogenesis of MSCs can typically require long culture periods that are impractical in a clinical setting and can lead to significant cost. Investigation into strategies that optimize cell production is essential. Here, we use the piezoelectric copolymer poly(vinylidene fluoride-trifluoroethylene) (PVDF-TrFE), functionalized with a poly(ethyl acrylate) (PEA) coating that drives fibronectin network formation, to enhance MSC adhesion and to present growth factors in the solid phase. Dynamic electrical cues are then incorporated, via a nanovibrational bioreactor, and the MSC response to electromechanical stimulation is investigated.

Questioning the foundations of the gut microbiota and obesity.

The role of the gut microbiota in determining body fatness has been a prominent area of research and has received significant public attention. Based largely on animal studies, recent attempts to translate these findings into interventions in humans have not been successful. This review will outline the key mouse research that initiated this area of study, examine whether those results warranted the initial enthusiasm and progress into human studies, and examine whether later follow-up research supported earlier conclusions. It will look at whether the absence of a gut microbiota protects germ-free mice from obesity, whether microbiota can transfer obesity into germ-free mice, the evidence for the role of immune system activation as a causal mechanism linking the gut microbiota to body weight, and consider the evidence for effects of individual bacterial species. Finally, it will examine the outcomes of randomized controlled trials of microbiota transfer in human participants that have not shown effects on body weight. With a more critical reading, early studies did not show as large an effect as first appeared and later research, including human trials, has failed to support a role of the gut microbiota in shaping body weight. This article is part of a discussion meeting issue 'Causes of obesity: theories, conjectures and evidence (Part II)'.

Biosynthesis of Zinc Oxide Nanoparticles on l-Carnosine Biofunctionalized Polyacrylonitrile Nanofibers; a Biomimetic Wound Healing Material.

Multifunctional biohybrid nanofibers (NFs) that can simultaneously drive various cellular activities and confer antibacterial properties are considered desirable in producing advanced wound healing materials. In this study, a bionanohybrid formulation was processed as a NF wound dressing to stimulate the adhesion and proliferation of fibroblast and endothelial cells that play a major role in wound healing. Polyacrylonitrile (PAN) electrospun NFs were hydrolyzed using NaOH and biofunctionalized with l-carnosine (CAR), a dipeptide which could later biosynthesize zinc oxide (ZnO) nanoparticles (NPs) on the NFs surface. The morphological study verified that ZnO NPs are uniformly distributed on the surface of CAR/PAN NFs. Through EDX and XRD analysis, it was validated that the NPs are composed of ZnO and/or ZnO/Zn(OH)2. The presence of CAR and ZnO NPs brought about a superhydrophilicity effect and notably raised the elastic modulus and tensile strength of Zn-CAR/PAN NFs. While CAR ligands were shown to improve the viability of fibroblast (L929) and endothelial (HUVEC) cells, ZnO NPs lowered the positive impact of CAR, most likely due to their repulsive negative surface charge. A scratch assay verified that CAR/PAN NFs and Zn-CAR/PAN NFs aided HUVEC migration more than PAN NFs. Also, an antibacterial assay implied that CAR/PAN NFs and Zn-CAR/PAN NFs are significantly more effective in inhibiting Staphylococcus aureus (S. aureus) than neat PAN NFs are (1000 and 500%, respectively). Taken together, compared to the neat PAN NFs, CAR/PAN NFs with and without the biosynthesized ZnO NPs can support the cellular activities of relevance for wound healing and inactivate bacteria.

Noninvasive Fecal Cytokine and Microbiota Profiles Predict Commencement of Necrotizing Enterocolitis in a Proof-of-Concept Study.

Background and Aims: Necrotizing enterocolitis (NEC) is a life-threatening disease and the most common gastrointestinal emergency in premature infants. Accurate early diagnosis is challenging. Modified Bell's staging is routinely used to guide diagnosis, but early diagnostic signs are nonspecific, potentially leading to unobserved disease progression, which is problematic given the often rapid deterioration observed. We investigated fecal cytokine levels, coupled with gut microbiota profiles, as a noninvasive method to discover specific NEC-associated signatures that can be applied as potential diagnostic markers. Methods: Premature babies born below 32 weeks of gestation were admitted to the 2-site neonatal intensive care unit (NICU) of Imperial College hospitals (St. Mary's or Queen Charlotte's & Chelsea) between January 2011 and December 2012. During the NICU stay, expert neonatologists grouped individuals by modified Bell's staging (healthy, NEC1, NEC2/3) and fecal samples from diapers were collected consecutively. Microbiota profiles were assessed by 16S rRNA gene amplicon sequencing and cytokine concentrations were measured by V-Plex multiplex assays. Results: Early evaluation of microbiota profiles revealed only minor differences. However, at later time points, significant changes in microbiota composition were observed for Bacillota (adj. P = .0396), with Enterococcus being the least abundant in Bell stage 2/3 NEC. Evaluation of fecal cytokine levels revealed significantly higher concentrations of IL-1α (P = .045), IL-5 (P = .0074), and IL-10 (P = .032) in Bell stage 1 NEC compared to healthy individuals. Conclusion: Differences in certain fecal cytokine profiles in patients with NEC indicate their potential use as diagnostic biomarkers to facilitate earlier diagnosis. Additionally, associations between microbial and cytokine profiles contribute to improving knowledge about NEC pathogenesis.

An efficient method for high molecular weight bacterial DNA extraction suitable for shotgun metagenomics from skin swabs.

The human skin microbiome represents a variety of complex microbial ecosystems that play a key role in host health. Molecular methods to study these communities have been developed but have been largely limited to low-throughput quantification and short amplicon-based sequencing, providing limited functional information about the communities present. Shotgun metagenomic sequencing has emerged as a preferred method for microbiome studies as it provides more comprehensive information about the species/strains present in a niche and the genes they encode. However, the relatively low bacterial biomass of skin, in comparison to other areas such as the gut microbiome, makes obtaining sufficient DNA for shotgun metagenomic sequencing challenging. Here we describe an optimised high-throughput method for extraction of high molecular weight DNA suitable for shotgun metagenomic sequencing. We validated the performance of the extraction method, and analysis pipeline on skin swabs collected from both adults and babies. The pipeline effectively characterised the bacterial skin microbiota with a cost and throughput suitable for larger longitudinal sets of samples. Application of this method will allow greater insights into community compositions and functional capabilities of the skin microbiome.

Engineered dual affinity protein fragments to bind collagen and capture growth factors.

Collagen type I lacks affinity for growth factors (GFs) and yet it is clinically used to deliver bone morphogenic protein 2 (BMP-2), a potent osteogenic growth factor. To mitigate this lack of affinity, supra-physiological concentrations of BMP-2 are loaded in collagen sponges leading to uncontrolled BMP-2 leakage out of the material. This has led to important adverse side effects such as carcinogenesis. Here, we design recombinant dual affinity protein fragments, produced in E. Coli, which contain two regions, one that spontaneously binds to collagen and a second one that binds BMP-2. By adding the fragment to collagen sponges, BMP-2 is sequestered enabling solid phase presentation of BMP-2. We demonstrate osteogenesis in vivo with ultra-low doses of BMP-2. Our protein technology enhances the biological activity of collagen without using complex chemistries or changing the manufacturing of the base material and so opens a pathway to clinical translation.

Fibronectin matrix assembly and TGFß1 presentation for chondrogenesis of patient derived pericytes for microtia repair.

Tissue engineered cartilage for external ear reconstruction of congenital deformities, such as microtia or resulting from trauma, remains a significant challenge for plastic and reconstructive surgeons. Current strategies involve harvesting autologous costal cartilage or expanding autologous chondrocytes ex vivo. However, these procedures often lead to donor site morbidity and a cell source with limited expansion capacity. Stromal stem cells such as perivascular stem cells (pericytes) offer an attractive alternative cell source, as they can be isolated from many human tissues, readily expanded in vitro and possess chondrogenic differentiation potential. Here, we successfully isolate CD146+ pericytes from the microtia remnant from patients undergoing reconstructive surgery (Microtia pericytes; MPs). Then we investigate their chondrogenic potential using the polymer poly(ethyl acrylate) (PEA) to unfold the extracellular matrix protein fibronectin (FN). FN unfolding exposes key growth factor (GF) and integrin binding sites on the molecule, allowing tethering of the chondrogenic GF transforming growth factor beta 1 (TGFß1). This system leads to solid-phase, matrix-bound, GF presentation in a more physiological-like manner than that of typical chondrogenic induction media (CM) formulations that tend to lead to off-target effects. This simple and controlled material-based approach demonstrates similar chondrogenic potential to CM, while minimising proclivity toward hypertrophy, without the need for complex induction media formulations.

Clinical NEC prevention practices drive different microbiome profiles and functional responses in the preterm intestine.

Preterm infants with very low birthweight are at serious risk for necrotizing enterocolitis. To functionally analyse the principles of three successful preventive NEC regimens, we characterize fecal samples of 55 infants (<1500 g, n = 383, female = 22) longitudinally (two weeks) with respect to gut microbiome profiles (bacteria, archaea, fungi, viruses; targeted 16S rRNA gene sequencing and shotgun metagenomics), microbial function, virulence factors, antibiotic resistances and metabolic profiles, including human milk oligosaccharides (HMOs) and short-chain fatty acids (German Registry of Clinical Trials, No.: DRKS00009290). Regimens including probiotic Bifidobacterium longum subsp. infantis NCDO 2203 supplementation affect microbiome development globally, pointing toward the genomic potential to convert HMOs. Engraftment of NCDO 2203 is associated with a substantial reduction of microbiome-associated antibiotic resistance as compared to regimens using probiotic Lactobacillus rhamnosus LCR 35 or no supplementation. Crucially, the beneficial effects of Bifidobacterium longum subsp. infantis NCDO 2203 supplementation depends on simultaneous feeding with HMOs. We demonstrate that preventive regimens have the highest impact on development and maturation of the gastrointestinal microbiome, enabling the establishment of a resilient microbial ecosystem that reduces pathogenic threats in at-risk preterm infants.

Keeping It Organized: Multicompartment Constructs to Mimic Tissue Heterogeneity.

Tissue engineering aims at replicating tissues and organs to develop applications in vivo and in vitro. In vivo, by engineering artificial constructs using functional materials and cells to provide both physiological form and function. In vitro, by engineering three-dimensional (3D) models to support drug discovery and enable understanding of fundamental biology. 3D culture constructs mimic cell-cell and cell-matrix interactions and use biomaterials seeking to increase the resemblance of engineered tissues with its in vivo homologues. Native tissues, however, include complex architectures, with compartmentalized regions of different properties containing different types of cells that can be captured by multicompartment constructs. Recent advances in fabrication technologies, such as micropatterning, microfluidics or 3D bioprinting, have enabled compartmentalized structures with defined compositions and properties that are essential in creating 3D cell-laden multiphasic complex architectures. This review focuses on advances in engineered multicompartment constructs that mimic tissue heterogeneity. It includes multiphasic 3D implantable scaffolds and in vitro models, including systems that incorporate different regions emulating in vivo tissues, highlighting the emergence and relevance of 3D bioprinting in the future of biological research and medicine.