Eben Alsberg

A method for forming a bioactive agent spatially patterned includes providing a photocrosslinkable hydrogel that includes a photocrosslinkable base polymer, photocrosslinkable bioactive agent coupling polymer macromers, and at least one bioactive agent that couples to the photocrosslinkable bioactive agent coupling polymer macromer, an selectively exposing discrete portions of the photocrosslinkable hydrogel to actinic radiation effective to initiate cross-linking of the base polymer and the bioactive agent coupling polymer macromers at the exposed portions.

Formation of chondromimetic human mesenchymal stem cells (hMSCs) condensations typically required in vitro culture in defined environments. In addition, extended in vitro culture in differentiation media over several weeks is usually necessary prior to implantation, which is costly, time consuming and delays clinical treatment. Here, this study reports on immediately implantable core/shell microgels with a high-density hMSC-laden core and rapidly degradable hydrogel shell. The hMSCs in the core formed cell condensates within 12 hours and the oxidized and methacrylated alginate (OMA) hydrogel shells were completely degraded within 3 days, enabling spontaneous and precipitous fusion of adjacent condensed aggregates. By delivering transforming growth factor-β1 (TGF-β1) within the core, the fused condensates were chondrogenically differentiated and formed cartilage microtissues. Importantly, these hMSC-laden core/shell microgels, fabricated without any in vitro culture, were subcutaneously implanted into mice and shown to form cartilage tissue via cellular condensations in the core after 3 weeks. This innovative approach to form cell condensations in situ without in vitro culture that can fuse together with each other and with host tissue and be matured into new tissue with incorporated bioactive signals, allows for immediate implantation and may be a platform strategy for cartilage regeneration and other tissue engineering applications.

HTTJABKRGRZYRN-UHFFFAOYSA-N Heparin Chemical compound OC1C (NC (= O) C) C (O) OC (COS (O)(= O)= O) C1OC1C (OS (O)(= O)= O) C (O) C (OC2C (C (OS (O)(= O)= O) C (OC3C (C (O) C (O) C (O3) C (O)= O) OS (O)(= O)= O) C (CO) O2) NS (O)(= O)= O) C (C (O)= O) O1 HTTJABKRGRZYRN-UHFFFAOYSA-N 0.000 description 5

Compromised vascular supply and insufficient neovascularization impede bone repair, increasing risk of non-union. Cyr61, Cysteine-rich angiogenic inducer of 61kD (also known as CCN1), is a matricellular growth factor that is regulated by mechanical cues during fracture repair. Here, we map the distribution of endogenous Cyr61 during bone repair and evaluate the effects of recombinant Cyr61 delivery on vascularized bone regeneration. In vitro, Cyr61 treatment did not alter chondrogenesis or osteogenic gene expression, but significantly enhanced angiogenesis. In a mouse femoral fracture model, Cyr61 delivery did not alter cartilage or bone formation, but accelerated neovascularization during fracture repair. Early initiation of ambulatory mechanical loading disrupted Cyr61-induced neovascularization. Together, these data indicate that Cyr61 delivery can enhance angiogenesis during bone repair, particularly for fractures with stable fixation, and may have therapeutic potential for fractures with limited blood vessel supply.

FHVDTGUDJYJELY-UHFFFAOYSA-N 6-{[2-carboxy-4, 5-dihydroxy-6-(phosphanyloxy) oxan-3-yl] oxy}-4, 5-dihydroxy-3-phosphanyloxane-2-carboxylic acid Chemical compound O1C (C (O)= O) C (P) C (O) C (O) C1OC1C (C (O)= O) OC (OP) C (O) C1O FHVDTGUDJYJELY-UHFFFAOYSA-N 0.000 description 23

The ability of cells to sense and respond to their physical environment plays a fundamental role in a broad spectrum of biological processes. As one of the most essential molecular force sensors and transducers found in cell membranes, mechanosensitive (MS) ion channels can convert mechanical inputs into biochemical or electrical signals to mediate a variety of sensations. The bottom-up construction of cell-sized compartments displaying cell-like organization, behaviors, and complexity, also known as synthetic cells, has gained popularity as an experimental platform to characterize biological functions in isolation. By reconstituting MS channels in the synthetic lipid bilayers, we envision using mechanosensitive synthetic cells for several medical applications. Here, we describe three different concepts for using ultrasound, shear stress, and compressive stress as mechanical stimuli to activate drug release from …

An interpenetrating polymer network (IPN) structured hydrogel includes a crosslinked first natural polymer macromer with a first elasticity and an interpenetrating network of crosslinked second natural polymer macromers having a second elasticity higher than the first elasticity, the IPN structured hydrogel being cytocompatible, and, upon degradation, produce substantially non-toxic products.

Human organoids have the potential to revolutionize in vitro disease modeling by providing multicellular architecture and function that are similar to those in vivo. This innovative and evolving technology, however, still suffers from assay throughput and reproducibility to enable high‐throughput screening (HTS) of compounds due to cumbersome organoid differentiation processes and difficulty in scale‐up and quality control. Using organoids for HTS is further challenged by the lack of easy‐to‐use fluidic systems that are compatible with relatively large organoids. Here, these challenges are overcome by engineering “microarray three‐dimensional (3D) bioprinting” technology and associated pillar and perfusion plates for human organoid culture and analysis. High‐precision, high‐throughput stem cell printing, and encapsulation techniques are demonstrated on a pillar plate, which is coupled with a complementary …

Poor nutrient transport through the cartilage endplate (CEP) is a key factor in the etiology of intervertebral disc degeneration and may hinder the efficacy of biologic strategies for disc regeneration. Yet, there are currently no treatments for improving nutrient transport through the CEP. In this study we tested whether intradiscal delivery of a matrix-modifying enzyme to the CEP improves solute transport into whole human and bovine discs. Ten human lumbar motion segments harvested from five fresh cadaveric spines (38–66 years old) and nine bovine coccygeal motion segments harvested from three adult steers were treated intradiscally either with collagenase enzyme or control buffer that was loaded in alginate carrier. Motion segments were then incubated for 18 h at 37 °C, the bony endplates removed, and the isolated discs were compressed under static (0.2 MPa) and cyclic (0.4–0.8 MPa, 0.2 Hz) loads while submerged in fluorescein tracer solution (376 Da; 0.1 mg/ml). Fluorescein concentrations from site-matched nucleus pulposus (NP) samples were compared between discs. CEP samples from each disc were digested and assayed for sulfated glycosaminoglycan (sGAG) and collagen contents. Results showed that enzymatic treatment of the CEP dramatically enhanced small solute transport into the disc. Discs with enzyme-treated CEPs had up to 10.8-fold (human) and 14.0-fold (bovine) higher fluorescein concentration in the NP compared to site-matched locations in discs with buffer-treated CEPs (p < 0.0001). Increases in solute transport were consistent with the effects of enzymatic treatment on CEP composition, which included …

A modular engineered tissue construct includes a plurality of fused self-assembled, scaffold-free, high-density cell aggregates. At least one cell aggregate includes a plurality of cells and a plurality of biocompatible and biodegradable nanoparticles and/or microparticles that are incorporated within the cell aggregates. The nanoparticles and/or microparticles acting as a bulking agent within the cell aggregate to increase the cell aggregate size and/or thickness and improve the mechanical properties of the cell aggregate as well as to deliver bioactive agents.

Within the complex microarchitecture of native cartilage tissue, the micromechanical properties of pericellular and extracellular matrices (PCM and ECM) potentially play important roles in developmental, physiological, and pathological processes. Here, we report a unique biomaterial-based engineering strategy to create cartilage-tissue equivalents possessing PCM-ECM microarchitecture of native cartilage, where human mesenchymal stem cell (hMSC)-laden soft microgels representing PCM are encapsulated in stiff hydrogels representing ECM. Mechanical property mismatches between soft PCM and stiff ECM under cyclic compression regulates hMSC proliferation and chondrogenesis. High PCM-ECM mechanical mismatch (softer PCM) and the presence of PCM degradation under cyclic compression individually or synergistically direct hMSC articular chondrogenesis through the proliferation-associated protein …

Oxygenating biomaterials can alleviate anoxic stress, stimulate vascularization, and improve engraftment of cellularized implants. However, the effects of oxygen-generating materials on tissue formation have remained largely unknown. Here, we investigate the impact of calcium peroxide (CPO)-based oxygen-generating microparticles (OMPs) on the osteogenic fate of human mesenchymal stem cells (hMSCs) under a severely oxygen deficient microenvironment. To this end, CPO is microencapsulated in polycaprolactone to generate OMPs with prolonged oxygen release. Gelatin methacryloyl (GelMA) hydrogels containing osteogenesis-inducing silicate nanoparticles (SNP hydrogels), OMPs (OMP hydrogels), or both SNP and OMP (SNP/OMP hydrogels) are engineered to comparatively study their effect on the osteogenic fate of hMSCs. OMP hydrogels associate with improved osteogenic differentiation under both …

Thermally stable or resilient proteins are usually stabilized at intermediate states during thermal stress to prevent irreversible denaturation. However, the mechanism by which their conformations are stabilized to resist high temperature remains elusive. Herein, we investigate the conformational and thermal stability of transforming growth factor-β1 (TGF-β1), a key signaling molecule in numerous biological pathways. We report that the TGF-β1 molecule is thermally resilient as it gradually denatures during thermal treatment when the temperature increases to 90°C–100°C but recovers native folding when the temperature decreases. Using this protein as a model, further studies show the maintenance of its bioactive functional properties after thermal stress, as demonstrated by differentiation induction of NIH/3T3 fibroblasts and human mesenchymal stem cells into myofibroblasts and chondrocytes, respectively …

The surface zone of articular cartilage is the first area impacted by cartilage defects, commonly resulting in osteoarthritis. Chondrocytes in the surface zone of articular cartilage synthesize and secrete lubricin, a proteoglycan that functions as a lubricant protecting the deeper layers from shear stress. Notably, 3D bioprinting is a tissue engineering technique that uses cells encapsulated in biomaterials to fabricate 3D constructs. Gelatin methacrylate (GelMA) is a frequently used biomaterial for 3D bioprinting cartilage. Oxidized methacrylated alginate (OMA) is a chemically modified alginate designed for its tunable degradation rate and mechanical properties. To determine an optimal combination of GelMA and OMA for lubricin expression, we used our novel high-throughput human articular chondrocyte reporter system. Primary human chondrocytes were transduced with PRG4 (lubricin) promoter-driven Gaussia luciferase, allowing for temporal assessment of lubricin expression. A lubricin expression-driven Design of Experiment screen and subsequent validation identified 14% GelMA/2% OMA for further study. Therefore, DoE optimized 14% GelMA/2% OMA, 14% GelMA control, and 16% GelMA (total solid content control) were 3D bioprinted. The combination of lubricin protein expression and shape retention over the 22 days in culture, successfully determined the 14% GelMA/2%OMA to be the optimal formulation for lubricin secretion. This strategy allows for rapid analysis of the role(s) of biomaterial composition, stiffness or other cell manipulations on lubricin expression by chondrocytes, which may improve therapeutic strategies for cartilage …

Advancements in 3D bioprinting have been hindered by the trade-off between printability and biological functionality. Existing bioinks struggle to meet both requirements simultaneously. However, new types of bioinks composed of densely packed microgels promise to address this challenge. These bioinks possess intrinsic porosity, allowing for cell growth, oxygen and nutrient transport, and better immunomodulatory properties, leading to superior biological functions. In this review, we highlight key trends in the development of these granular bioinks. Using examples, we demonstrate how granular bioinks overcome the trade-off between printability and cell function. Granular bioinks show promise in 3D bioprinting, yet understanding their unique structure–property–function relationships is crucial to fully leverage the transformative capabilities of these new types of bioinks in bioprinting.

In human vascular anatomy, blood flows from the heart to organs and tissues through a hierarchical vascular tree, comprising large arteries that branch into arterioles and further into capillaries, where gas and nutrient exchange occur. Engineering a complete, integrated vascular hierarchy with vessels large enough to suture, strong enough to withstand hemodynamic forces, and a branching structure to permit immediate perfusion of a fluidic circuit across scales would be transformative for regenerative medicine (RM), enabling the translation of engineered tissues of clinically relevant size, and perhaps whole organs. How close are we to solving this biological plumbing problem? In this review, we highlight advances in engineered vasculature at individual scales and focus on recent strategies to integrate across scales.

Stimuli‐responsive hydrogels are intriguing biomimetic materials. Previous efforts to develop mechano‐responsive hydrogels have mostly relied on chemical modifications of the hydrogel structures. Here, we present a simple, generalizable strategy that confers mechano‐responsive behavior on hydrogels. Our approach involves embedding hybrid vesicles, composed of phospholipids and amphiphilic block copolymers, within the hydrogel matrix to act as signal transducers. Under mechanical stress, these vesicles undergo deformation and rupture, releasing encapsulated compounds that can control the hydrogel network. To demonstrate this concept, we embedded vesicles containing ethylene glycol tetraacetic acid (EGTA), a calcium chelator, into a calcium‐crosslinked alginate hydrogel. When compressed, the released EGTA sequesters calcium ions and degrades the hydrogel. This study provides a novel …

Corneal injuries are a major cause of blindness worldwide. To restore corneal integrity and clarity, there is a need for regenerative biointegrating materials for in situ repair and replacement of corneal tissue. Here, light‐curable cornea matrix (LC‐COMatrix), a tunable material derived from decellularized porcine cornea extracellular matrix containing un‐denatured collagen and sulfated glycosaminoglycans is introduced. It is a functionalized hydrogel with proper swelling behavior, biodegradation, and viscosity that can be cross‐linked in situ with visible light, providing significantly enhanced biomechanical strength, stability, and adhesiveness. The cross‐linked LC‐COMatrix strongly adheres to human corneas ex vivo and effectively closes full‐thickness corneal perforations with tissue loss. Likewise, in vivo, LC‐COMatrix seals large corneal perforations, replaces partial‐corneal stromal defects and biointegrates into …

A method of forming a gel particle slurry includes providing a first solution that includes a cross-linkable hydrogel polymer macromer and an optional first crosslinker in a first depot and optionally a second solution in a second depot that is separated from the first depot by a mixing unit that includes a mixing element; and reversibly transferring the first solution and the optional second solution through the mixing unit between the first depot and the second depot such that the first solution and the optional second solution are mixed and agitated to form the gel particle slurry.

For bioprinting of 4D living tissues, in article number 2109394, Eben Alsberg and co-workers devise a cell-laden bioink featuring high-resolution printing, physiological-trigger-enabled shape morphing, and long-term cell viability and function. With this system, they demonstrate that the printed cell-rich bioconstructs can exert multidirectional reshaping in a controlled manner and transform and develop into tissues with sophisticated structures. The system is anticipated to advance bioprinting to a horizon that enables 4D biomimetic tissue engineering.