Hematopoietic stem cells (HSCs) play a crucial role in the generation of the body’s blood and immune cells. This process takes place primarily in the bone marrow in specialized ‘niche’ microenvironments, which provide signals responsible for maintaining a balance between HSC quiescence, self-renewal, and lineage specification required for life-long hematopoiesis. While our understanding of these signaling mechanisms continues to improve, our ability to engineer them in vitro for the expansion of clinically relevant HSC populations is still lacking. In this review, we focus on development of biomaterials-based culture platforms for in vitro study of interactions between HSCs and their local microenvironment. The tools and techniques used for both examining HSC-niche interactions as well as applying these findings towards controlled HSC expansion or directed differentiation in 2D and 3D platforms are discussed. These novel techniques hold the potential to push the existing boundaries of HSC cultures towards high-throughput, real-time, and single-cell level biomimetic approaches that enable a more nuanced understanding of HSC regulation and function. Their application in conjunction with innovative biomaterial platforms can pave the way for engineering artificial bone marrow niches for clinical applications as well as elucidating the pathology of blood-related cancers and disorders.

Vascularization is among the top challenges that impede the clinical application of engineered tissues. This challenge has spurred tremendous research endeavor, defined as vascular tissue engineering (VTE) in this article, to establish a pre-existing vascular network inside the tissue engineered graft prior to implantation. Ideally, the engineered vasculature can be integrated into the host vasculature via anastomosis to supply nutrient to all cells instantaneously after surgery. Moreover, sufficient vascularization is of great significance in regenerative medicine from many other perspectives. Due to the critical role of vascularization in successful tissue engineering, we aim to provide an up-to-date overview of the fundamentals and VTE strategies in this article, including angiogenic cells, biomaterial/bio-scaffold design and bio-fabrication approaches, along with the reported utility of vascularized tissue complex in regenerative medicine. We will also share our opinion on the future perspective of this field.

Hematopoietic stem cells (HSCs) are a rare stem cell population found primarily in the bone marrow and responsible for the production of the body’s full complement of blood and immune cells. Used clinically to treat a range of hematopoietic disorders, there is a significant need to identify approaches to selectively expand their numbers ex vivo. Here we describe a methacrylamide-functionalized gelatin (GelMA) hydrogel for in vitro culture of primary murine HSCs. Stem cell factor (SCF) is a critical biomolecular component of native HSC niches in vivo and is used in large dosages in cell culture media for HSC expansion in vitro. We report a photochemistry based approach to covalently immobilize SCF within GelMA hydrogels via acrylate-functionalized polyethylene glycol (PEG) tethers. PEG-functionalized SCF retains the native bioactivity of SCF but can be stably incorporated and retained within the GelMA hydrogel over 7 days. Freshly-isolated murine HSCs cultured in GelMA hydrogels containing covalently-immobilized SCF showed reduced proliferation and improved selectivity for maintaining primitive HSCs. Comparatively, soluble SCF within the GelMA hydrogel network induced increased proliferation of differentiating hematopoietic cells. We used a microfluidic templating approach to create GelMA hydrogels containing gradients of immobilized SCF that locally direct HSC response. Together, we report a biomaterial platform to examine the effect of the local presentation of soluble vs. matrix-immobilized biomolecular signals on HSC expansion and lineage specification. This approach may be a critical component of a biomaterial-based artificial bone marrow to provide the correct sequence of niche signals to grow HSCs in the laboratory.

The skin is responsible for several important physiological functions and has enormous clinical significance in wound healing. Tissue engineered substitutes may be used in patients suffering from skin injuries to support regeneration of the epidermis, dermis, or both. Skin substitutes are also gaining traction in the cosmetics and pharmaceutical industries as alternatives to animal models for product testing. Recent biomedical advances, ranging from cellular‐level therapies such as mesenchymal stem cell or growth factor delivery, to large‐scale biofabrication techniques including 3D printing, have enabled the implementation of unique strategies and novel biomaterials to recapitulate the biological, architectural, and functional complexity of native skin. This progress report highlights some of the latest approaches to skin regeneration and biofabrication using tissue engineering techniques. Current challenges in fabricating multilayered skin are addressed, and perspectives on efforts and strategies to meet those limitations are provided. Commercially available skin substitute technologies are also examined, and strategies to recapitulate native physiology, the role of regulatory agencies in supporting translation, as well as current clinical needs, are reviewed. By considering each of these perspectives while moving from bench to bedside, tissue engineering may be leveraged to create improved skin substitutes for both in vitro testing and clinical applications.

The bone marrow provides spatially and temporally variable signals that impact the behavior of hematopoietic stem cells (HSCs). While multiple biomolecular signals and bone marrow cell populations have been proposed as key regulators of HSC fate, new tools are required to probe their importance and mechanisms of action. Here, a novel method based on a microfluidic mixing platform to create small volume, 3D hydrogel constructs containing overlapping patterns of cell and matrix constituents inspired by the HSC niche is described. This approach is used to generate hydrogels containing opposing gradients of fluorescent microspheres, MC3T3-E1 osteoblasts, primary murine hematopoietic stem and progenitor cells (HSPCs), and combinations thereof in a manner independent of hydrogel density and cell/particle size. Three different analytical methods are described to characterize local properties of these hydrogels at multiple scales: 1) whole construct fluorescent analysis; 2) multi-photon imaging of individual cells within the construct; 3) retrieval of discrete sub-regions from the hydrogel post-culture. The approach reported here allows the creation of stable gradients of cell and material cues within a single, optically translucent 3D biomaterial to enable a range of investigations regarding how microenvironmental signals impact cell fate.

Hematopoietic stem cells (HSC) reside in unique bone marrow niches and are influenced by signals from surrounding cells, the extracellular matrix (ECM), ECM-bound or diffusible biomolecules. Here we describe the use of a three-dimensional hydrogel to alter the balance of HSC-generated autocrine feedback and paracrine signals generated by co-cultured niche-associated cells. We report shifts in HSC proliferation rate and fate specification in the presence of lineage positive (Lin+) niche cells. Hydrogels promoting autocrine feedback enhanced expansion of early hematopoietic progenitors while paracrine signals from Lin+ cells increased myeloid differentiation. We report thresholds where autocrine vs. paracrine cues alter HSC fate transitions, and were able to selectively abrogate the effects of matrix diffusivity and niche cell co-culture via the use of inhibitory cocktails of autocrine or paracrine signals. Together, these results suggest diffusive biotransport in three-dimensional biomaterials are a critical design element for the development of a synthetic stem cell niche.

As the most versatile and promising cell source, stem cells have been studied in regenerative medicine for two decades. Currently available culturing techniques utilize a 2D or 3D microenvironment for supporting the growth and proliferation of stem cells. However, these culture systems fail to fully reflect the supportive biological environment in which stem cells reside in vivo, which contain dynamic biophysical growth cues. Herein, a 4D programmable culture substrate with a self‐morphing capability is presented as a means to enhance dynamic cell growth and induce differentiation of stem cells. To function as a model system, a 4D neural culture substrate is fabricated using a combination of printing and imprinting techniques keyed to the different biological features of neural stem cells (NSCs) at different differentiation stages. Results show the 4D culture substrate demonstrates a time‐dependent self‐morphing process that plays an essential role in regulating NSC behaviors in a spatiotemporal manner and enhances neural differentiation of NSCs along with significant axonal alignment. This study of a customized, dynamic substrate revolutionizes current stem cell therapies, and can further have a far‐reaching impact on improving tissue regeneration and mimicking specific disease progression, as well as other impacts on materials and life science research.

Despite recent advances in clinical procedures, the repair of soft tissue remains a reconstructive challenge. Current technologies such as synthetic implants and dermal flap autografting result in inefficient shape retention and unpredictable aesthetic outcomes. 3D printing, however, can be leveraged to produce superior soft tissue grafts that allow enhanced host integration and volume retention. Here, a novel dual bioink 3D printing strategy is presented that utilizes synthetic and natural materials to create stable, biomimetic soft tissue constructs. A double network ink composed of covalently crosslinked poly(ethylene) glycol and ionically crosslinked alginate acts as a physical support network that promotes cell growth and enables long-tersm graft shape retention. This is coupled with a cell-laden, biodegradable gelatin methacrylate bioink in a hybrid printing technique, and the composite scaffolds are evaluated in their mechanical properties, shape retention, and cytotoxicity. Additionally, a new shape analysis technique utilizing CloudCompare software is developed that expands the available toolbox for assessing scaffold aesthetic properties. With this dynamic 3D bioprinting strategy, complex geometries with robust internal structures can be easily modulated by varying the print ratio of non-degradable to sacrificial strands. The versatility of this hybrid printing fabrication platform can inspire the design of future multi-material regenerative implants.