Architectural Cues Research Articles

Current Methodologies for Inducing Aligned Myofibers in Tissue Constructs for Skeletal Muscle Tissue Regeneration.

Significance: Volumetric muscle loss (VML) results in the loss of large amounts of tissue that inhibits muscle regeneration. Existing therapies, such as autologous muscle transfer and physical therapy, are incapable of returning full function and force production to injured muscle. Recent Advances: Skeletal muscle tissue constructs may provide an alternative to existing therapies currently used to treat VML. Unlike autologous muscle transplants, muscle constructs can be cultured in vitro and are not reliant on intact muscle tissue. Skeletal muscle constructs can be generated from small muscle biopsies and could be used to generate skeletal muscle tissue constructs to replace injured tissues. Critical Issues: To serve as effective therapies, muscle constructs must be capable of generating contractile forces that can assist the function of host skeletal muscle. The contractile force of native muscle arises in part as a consequence of the highly aligned, bundled architecture of myofibers. Attempts to induce similar alignment include applications of tension/strain across hydrogels, inducing aligned architectures within scaffolds, casting tissues in straited molds, and 3D printing. While all these methods have demonstrated efficacy toward inducing myofiber alignment, the extent of myofiber alignment, tissue formation, and force production varies. This manusript critically reviews the advantages and limitations of these methods and specifically discusses their ability to impart mechanical and architectural cues to induce alignment within tissue constructs. Future Directions: As tissue-synthesizing techniques continue to improve, muscle constructs must include more cell types than simply myoblasts, such as the addition of neuronal and endothelial cells. Higher-level tissue organization is critical to the success of these constructs. Many of these technologies have yet to be implanted into host tissue to understand engraftment and how they can contribute to traumatic injury, and as such continued collaboration between surgeons and tissue engineers is necessary to ultimately result in clinical translation.

Glutenin-chitosan 3D porous scaffolds with tunable stiffness and systematized microstructure for cultured meat model

A glutenin (G)-chitosan (CS) complex (G-CS) was cross-linked by water annealing with aim to prepare structured 3D porous cultured meat scaffolds (CMS) here. The CMS has pore diameters ranging from 18 to 67 μm and compressive moduli from 16.09 to 60.35 kPa, along with the mixing ratio of G/CS. SEM showed the porous organized structure of CMS. FTIR and CD showed the increscent content of α-helix and β-sheet of G and strengthened hydrogen-bondings among G-CS molecules, which strengthened the stiffness of G-CS. Raman spectra exhibited an increase of G concentration resulted in higher crosslinking of disulfide-bonds in G-CS, which aggrandized the bridging effect of G-CS and maintained its three-dimensional network. Cell viability assay and immuno-fluorescence staining showed that G-CS effectively facilitated the growth and myogenic differentiation of porcine skeletal muscle satellite cells (PSCs). CLSM displayed that cells first occupied the angular space of hexagon and then ring-growth circle of PSCs were orderly formed on G-CS. The texture and color of CMS which loaded proliferated PSCs were fresh-meat like. These results showed that physical cross-linked G-CS scaffolds are the biocompatible and stable adaptable extracellular matrix with appropriate architectural cues and natural micro-environment for structured CM models.

Investigating expert and lay judgments of pathogen transmission risk in urban and architectural environments

ABSTRACT The threat of infection posed by the COVID-19 virus forced the general public to use the visible cues within urban and architectural environments as a ‘lens’ through which pathogen transmission risk could be inferred. This study presents a model that quantifies associations between (1) the visible cues of urban and architectural environments and lay ratings of pathogen transmission risk, (2) the same relationship for experts (i.e. Infection Control Practitioners), and (3) the association between the lay ratings and the expert ratings. A series of urban and architectural environments were rated on twenty visible architectural cues and for their perceived pathogen transmission risk by lay and expert raters. Correlational analyses between the two groups yielded considerable consensus between risk ratings, as well as between which cues were significantly associated with risk ratings, which included the space’s crowdedness, the potential for crowds, and cleanliness. Expert risk ratings were also significantly associated with corridor size, and marginally significantly associated with the number of touchable surfaces, the number of furniture/seating, and access to fresh air. In this way, expert cue utilization is more complex than lay assessments. Implications for public health policy makers and designers of the built environment are discussed.

Unravelling microRNA regulation and miRNA-mRNA regulatory networks in osteogenesis driven by 3D nanotopographical cues.

Three-dimensional (3D) culturing of cells is being adopted for developing tissues for various applications such as mechanistic studies, drug testing, tissue regeneration, and animal-free meat. These approaches often involve cost-effective differentiation of stem or progenitor cells. One approach is to exploit architectural cues on a 3D substrate to drive cellular differentiation, which has been shown to be effective in various studies. Although extensive gene expression data from such studies have shown that gene expression patterns might differ, the gene regulatory networks controlling the expression of genes are rarely studied. In this study, we profiled genes and microRNAs (miRNAs) via next-generation sequencing (NGS) in human mesenchymal stem cells (hMSCs) driven toward osteogenesis via architectural cues in 3D matrices (3D conditions) and compared with cells in two-dimensional (2D) culture driven toward osteogenesis via soluble osteoinductive factors (OF conditions). The total number of differentially expressed genes was smaller in 3D compared to OF conditions. A distinct set of genes was observed under these conditions that have been shown to control osteogenic differentiation via different pathways. Small RNA sequencing revealed a core set of miRNAs to be differentially expressed under these conditions, similar to those that have been previously implicated in osteogenesis. We also observed a distinct regulation of miRNAs in these samples that can modulate gene expression, suggesting supplementary gene regulatory networks operative under different stimuli. This study provides insights into studying gene regulatory networks for identifying critical nodes to target for enhanced cellular differentiation and reveal the differences in physical and biochemical cues to drive cell fates.

3D printed hydrogel/bioceramics core/shell scaffold with NIR-II triggered drug release for chemo-photothermal therapy of bone tumors and enhanced bone repair

The treatment of bone defect caused by osteosarcoma remains an intractable problem. Scaffolds with multi-functions are the potential candidates to kill recurrent tumor and to repair the bone defects after tumorectomy. However, most of these scaffolds employed photothermal therapy using near‐infrared laser inthe first biological window (NIR‐I)with intrinsic poor tissue penetration depth. Some scaffolds loading drugs for chemotherapy generally lack the accurate control of on-demand drugs release to reduce the side effects and enhance the therapeutic efficacy. Herein, 3D printed gelatin/bioceramics core/shell scaffolds were developed with doxorubicin (DOX) loaded gelatin as the core part and SrCuSi4O10 (SC) nanosheets/beta-tricalcium phosphate (β-TCP) as the shell part of the printed filaments. SC nanosheets endowed the scaffolds with photothermal therapy under NIR-IIlaser irradiation. Simultaneously, the generated extensive hyperthermia could induce the gel-sol transition of the gelatin in the core part of the filaments, which subsequently triggered the on-demand DOX release from the loosened gelatin achieving chemo-photothermal therapy. The in vitro and in vivo data demonstrated that chemo-photothermal therapy showed a synergetic effect on anti-tumor. Additionally, the degradation/release of gelatin from the filaments resulted in the hollow channels in the scaffold, which provided clear architectural cues for promoting the ingrowth of bone tissues. Meanwhile, the degradation of the SC nanosheets contributed to the sustained release of bioactive ions (Sr, Cu and Si), which further enhanced vascularized bone regeneration. All these results indicated that the 3D printed DOX loaded gelatin-TCP/SC scaffolds had a great potential application for the treatment of bone defects caused by osteosarcoma in effective killing cancer cells and repairing tumor-induced bone defects.

Programming Delayed Dissolution Into Sacrificial Bioinks For Dynamic Temporal Control of Architecture within 3D‐Bioprinted Constructs (Adv. Funct. Mater. 8/2023)

Bioprinting In article number 2210521, Khoon S. Lim, and co-workers highlight a scaffolding approach to engineering blood vessels, starting from lego like construction of macro-scale vessels to functional micro-scale vessels. Programmable sacrificial bioinks are developed, and applied to a range of biofabrication platforms, introducing architectural cues into thick hydrogel constructs in a spatial-temporal manner, to direct cell function and alignment.

Programming Delayed Dissolution Into Sacrificial Bioinks For Dynamic Temporal Control of Architecture within 3D‐Bioprinted Constructs

AbstractSacrificial printing allows introduction of architectural cues within engineered tissue constructs. This strategy adopts the use of a 3D‐printed sacrificial ink that is embedded within a bulk hydrogel which is subsequently dissolved to leave open‐channels. However, current conventional sacrificial inks do not recapitulate the dynamic nature of tissue development, such as the temporal presentation of architectural cues matching cellular requirements during different stages of maturation. To address this limitation, a new class of sacrificial inks is developed that exhibits tailorable and programmable delayed dissolution profiles (1–17 days), by exploiting the unique ability of the ruthenium complex and sodium persulfate initiating system to crosslink native tyrosine groups present in non‐chemically modified gelatin. These novel sacrificial inks are also shown to be compatible with a range of biofabrication technologies, including extrusion‐based printing, digital‐light processing, and volumetric bioprinting. Further embedding these sacrificial templates within cell‐laden bulk hydrogels displays precise control over the spatial and temporal introduction of architectural features into cell‐laden hydrogel constructs. This approach demonstrates the unique capacity of delaying dissolution of sacrificial inks to modulate cell behavior, improving the deposition of mineralized matrix and capillary‐like network formation in osteogenic and vasculogenic culture, respectively.

Accelerated vascularization of a novel collagen hydrogel dermal template.

Full thickness skin loss is a debilitating problem, most commonly reconstructed using split thickness skin grafts (STSG), which do not reconstitute normal skin thickness and often result in suboptimal functional and esthetic outcomes that diminish a patient's quality of life. To address the minimal dermis present in most STSG, engineered dermal templates were developed that can induce tissue ingrowth and the formation of neodermal tissue. However, clinically available dermal templates have many shortcomings including a relatively slow rate and degree of neovascularization (∼2-4weeks), resulting in multiple dressing changes, prolonged immobilization, and susceptibility to infection. Presented herein is a novel composite hydrogel scaffold that optimizes a unique scaffold microarchitecture with native hydrogel properties and mechanical cues ideal for promoting neovascularization, tissue regeneration, and wound healing. In vitro analysis demonstrated the unique combination of improved mechanical attributes with native hydrogel properties that promotes cell invasion and remodeling within the scaffold. In a novel 2-stage rat model of full thickness skin loss that closely mimics clinical practice, the composite hydrogel induced rapid cell infiltration and neovascularization, creating a healthy neodermis after only 1week onto which a skin graft could be placed. The scaffold also elicited a gradual and favorable immune response, resulting in more efficient integration into the host. We have developed a dermal scaffold that utilizes simple but unique collagen hydrogel architectural cues that rapidly induces the formation of stable, functional neodermal tissue, which holds tremendous promise for the treatment of full thickness skin loss.

3D printed hydrogel/wesselsite-PCL composite scaffold with structural change from core/shell fibers to microchannels for enhanced bone regeneration

Corresponding to the process of bone formation, three-dimensional (3D) scaffolds should provide adaptive structural cues and microenvironment for bone repair at different stages. Herein, we fabricate a core-shell fiber scaffold by coating a layer of polycaprolactone (PCL)/SrCuSi4O10 (wesselsite, CS) on 3D printed hydrogel scaffolds. The core/shell scaffold provided enhanced mechanical support for the defect at the initial stage. Then, the degradation of hydrogels resulted in the transformation of core/shell fiber structure (hydrogel/PCL + CS) to interconnected microchannels (PCL/CS hollow channel), which provided increasing space and clear architectural cues for promoting bone formation and vascularization. Simultaneously, the chemokine stromal cell-derived factor-1α (SDF-1α) loaded in hydrogels achieved sustained release in the initial two weeks to facilitate the recruitment of bone marrow stromal cells (BMSCs) to defect. Subsequently, the degradation of CS nanomaterials yielded the sustained release of bioactive ions (Si, Sr and Cu), promoting osteogenesis and angiogenesis. The in vivo data showed that the prepared scaffolds exhibited enhanced bone repair capacity with abundant new bone formation and blood vessels ingrowth in the defect. Thus, this designed system shows great potential for bone tissue engineering application.

Synthetic vascular graft with spatially distinct architecture for rapid biomimetic cell organisation in a perfusion bioreactor

Access to lab-grown fully functional blood vessels would provide an invaluable resource to vascular medicine. The complex architecture and cellular makeup of native vessels, however, makes this extremely challenging to reproduce in vitro. Bioreactor systems have helped advanced research in this area by replicating many of the physiological conditions necessary for full-scale tissue growth outside of the body. A key element underpinning these technologies are 3D vascular graft templates which serve as temporary scaffolds to direct cell growth into similar cellular architectures observed in native vessels. Grafts further engineered with appropriate physical cues to accommodate the multiple cell types that reside within native vessels may help improve the production efficiency and physiological accuracy of bioreactor-grown vessel substitutes. Here, we engineered two distinct scaffold architectures into an electrospun vascular graft aiming to encourage the spatial organisation of human vascular endothelial cells (hCAECs) in a continuous luminal monolayer, co-cultured with human fibroblasts (hFBs) populating the graft wall. Using an electrospun composite of polycaprolactone and gelatin, we evaluated physical parameters including fibre diameter, fibre alignment, and porosity, that best mimicked the spatial composition and growth of hCAECs and hFBs in native vessels. Upon identifying the optimal scaffold architectures for each cell type, we constructed a custom-designed mandrel that combined these distinct architectures into a single vascular graft during a single electrospinning processing run. When connected to a perfusion bioreactor system, the dual architecture graft spatially oriented hCAECs and hFBs into the graft wall and lumen, respectively, directly from circulation. This biomimetic cell organisation was consistent with positive graft remodelling with significant collagen deposition in the graft wall. These findings demonstrate the influence of architectural cues to direct cell growth within vascular graft templates and the future potential of these approaches to more accurately and efficiency produce blood vessel substitutes in bioreactor systems.

Current Strategies for the Regeneration of Skeletal Muscle Tissue.

Traumatic injuries, tumor resections, and degenerative diseases can damage skeletal muscle and lead to functional impairment and severe disability. Skeletal muscle regeneration is a complex process that depends on various cell types, signaling molecules, architectural cues, and physicochemical properties to be successful. To promote muscle repair and regeneration, various strategies for skeletal muscle tissue engineering have been developed in the last decades. However, there is still a high demand for the development of new methods and materials that promote skeletal muscle repair and functional regeneration to bring approaches closer to therapies in the clinic that structurally and functionally repair muscle. The combination of stem cells, biomaterials, and biomolecules is used to induce skeletal muscle regeneration. In this review, we provide an overview of different cell types used to treat skeletal muscle injury, highlight current strategies in biomaterial-based approaches, the importance of topography for the successful creation of functional striated muscle fibers, and discuss novel methods for muscle regeneration and challenges for their future clinical implementation.

Microchannels Are an Architectural Cue That Promotes Integration and Vascularization of Silk Biomaterials in Vivo.

Functional integration of implanted biomaterials and bioengineered tissues in vivo requires effective and timely vascular ingrowth. While many vascularization strategies rely on delivery of angiogenic growth factors or endothelial cells to promote vascular ingrowth, the effect of physical and architectural features of biomaterials on the vascularization process is less well understood. Microchannels are a simple, accessible architectural feature frequently engineered into 3D biomaterials to promote mass transfer. In this study, the effect of microchannels on the integration and vascularization of 3D porous silk scaffolds was explored over a 14 week period. An array of 508 μm diameter microchannels spanning the length of critically sized, porous silk scaffolds significantly improved tissue ingrowth into the constructs. At week 6, all silk scaffolds (n = 8) with microchannels showed complete tissue infiltration throughout the construct, while only one of eight (12.5%) did so in the absence of microchannels. The presence of microchannels improved silk scaffold vascularization with significantly more vessels per unit area in the presence of microchannels. The vessel size distribution was similar in both scaffold types, but a shift in distribution toward smaller vessels was observed in the presence of microchannels. The blood vessels in silk scaffolds were perfused, functional and connected to the animal's cardiovascular system, as demonstrated by the presence of red blood cells in the vessel lumens, and effective delivery of a contrast agent the vessels inside the scaffold. This study demonstrates the utility of microchannels as a simple architectural feature that significantly improves vascularization and integration of implanted biomaterials.

Graft alignment impacts the regenerative response of skeletal muscle after volumetric muscle loss in a rat model

A key event in the etiology of volumetric muscle loss (VML) injury is the bulk loss of structural cues provided by the underlying extracellular matrix (ECM). To re-establish the lost cues, there is broad consensus within the literature supporting the utilization of implantable scaffolding. However, while scaffold based regenerative medicine strategies have shown potential, there remains a significant amount of outcome variability observed across the field. We suggest that an overlooked source of outcome variability is differences in scaffolding architecture. The goal of this study was to test the hypothesis that implant alignment has a significant impact on genotypic and phenotypic outcomes following the repair of VML injuries. Using a rat VML model, outcomes across three autograft implant treatment groups (aligned implants, 45° misaligned, and 90° misaligned) and two recovery time points (2 weeks and 12 weeks) were examined (n = 6–8/group). At 2 weeks post-repair there were no significant differences in muscle mass and torque recovery between the treatment groups, however we did observe a significant upregulation of MyoD (2.5 fold increase) and Pax7 (2 fold increase) gene expression as well as the presence of immature myofibers at the implant site for those animals repaired with aligned autografts. By 12 weeks post-repair, functional and structural differences between the treatment groups could be detected. Aligned autografts had significantly greater mass and torque recovery (77 ± 10% of normal) when compared to 45° and 90° misaligned autografts (64 ± 10% and 61 ± 11%, respectively). Examination of tissue structure revealed extensive fibrosis and a significant increase in non-contractile tissue area fraction for only those animals treated using misaligned autografts. When taken together, the results suggest that implant graft orientation has a significant impact on in-vivo outcomes and indicate that the effect of graft alignment on muscle phenotype may be mediated through genotypic changes to myogenesis and fibrosis at the site of injury and repair. Statement of SignificanceA key event in the etiology of volumetric muscle loss injury is the bulk loss of architectural cues provided by the underlying extracellular matrix. To re-establish the lost cues, there is broad consensus within the literature supporting the utilization of implantable scaffolding. Yet, although native muscle is a highly organized tissue with network and cellular alignment in the direction of contraction, there is little evidence within the field concerning the importance of re-establishing native architectural alignment. The results of this study suggest that critical interactions exist between implant and native muscle alignment cues during healing, which influence the balance between myogenesis and fibrosis. Specifically, it appears that alignment of implant architectural cues with native muscle cues is necessary to create a pro-myogenic environment and contractile force recovery. The results also suggest that misaligned cues may be pathological, leading to fibrosis and poor contractile force recovery.

Exploring staircases as architectural cues in virtual vertical navigation

• Local architectural cues are seen as salient landmarks. • Staircases enable vertical circulation in multi-level buildings. • Staircase attributes influence virtual vertical navigation behavior. • Virtual navigation and individual differences affect staircase preferences. • Interior design heuristics are provided for staircases. Architectural design requires experiencing the spatial organization of a building, discovering architectural cues and maintaining spatial orientation during navigation. Architects configure architectural cues in the initial phase of the design process. Staircases, as a feature of local architectural cues that provide access to the other floors in a multi-level building, can have an impact on vertical navigation and aid individuals during navigation and influence their spatial orientation. This study focuses on the issue of vertical navigation during virtual navigation by integrating the individual differences and the geometric attributes of a staircase pair within two different multi-level desktop virtual environments (VEs). The angle between the cue pairs with respect to the same observation point is altered in order to determine the staircase pair that is more efficient in navigation. Virtual vertical navigation is based on an egocentric frame of reference where the participants have control of their movements. Circulation paths, gender differences, navigational abilities and cue pairs are the factors that affect staircase preferences for ascending and descending. For the VE with a 180° difference between the cue pairs, a relationship was found between the ascending and descending staircases. Further analysis indicated that the staircase preference in ascending was either related to the first or last visited rooms on the ground floor. For the VE with a 90° difference between the cue pairs, no relationship was found between the ascending and descending staircases as well as with any other factor. There was only a significant relationship between gender and staircase preference in descending staircases with 180° difference between the cue pairs in favor of females. In addition, there was no significant relationship between the navigational abilities and staircase preferences.

Tissue Architectural Cues Drive Organ Targeting of Tumor Cells in Zebrafish.

Tumor cells encounter a myriad of physical cues upon arrest and extravasation in capillary beds. Here, we examined the role of physical factors in non-random organ colonization using a zebrafish xenograft model. We observed a two-step process by which mammalian mammary tumor cells showed non-random organ colonization. Initial homing was driven by vessel architecture, where greater numbers of cells became arrested in the topographically disordered blood vessels of the caudal vascular plexus (CVP) than in the linear vessels in the brain. Following arrest, bone-marrow- and brain-tropic clones exhibited organ-specific patterns of extravasation. Extravasation was mediated by β1 integrin, where knockdown of β1 integrin reduced extravasation in the CVP but did not affect extravasation of a brain-tropic clone in the brain. In contrast, silencing myosin 1B redirected early colonization from the brain to the CVP. Our results suggest that organ selectivity is driven by both vessel topography and cell-type-dependent extravasation.

Influence of fiber architecture and growth factor formulation on osteoblastic differentiation of mesenchymal stem cells in coacervate-coated electrospun fibrous scaffolds

In order to achieve effective bone regeneration, the architectural/structural and biological environment must be considered. In this study, we investigated the influence of structure of extracellular matrix-mimicking electrospun poly (l-lactic acid) (PLLA) fibrous scaffolds and incorporated growth factors (bone morphogenetic protein2 (BMP2) and platelet-derived growth factor (PDGF)) (GFs) on in vitro differentiation of human mesenchymal stem cells (hMSCs) cultured onto the scaffolds. Furthermore, Cargo GFs were first encapsulated into coacervate (Coa). Coa-coated nano-sized PLLA (Coa-nPLLA) exhibited relatively higher coating efficacy, than Coa-coated micro-sized PLLA (Coa-mPLLA) due to large mesh hole area of mPLLA. Consequently, a faster GF release pattern was observed in nPLLA groups. As a result of osteogenic differentiation of hMSC which cultured onto GFs loaded Coa-PLLA, the architectural cue of fiber diameters effectively modulated early osteogenic differentiation of hMSCs. Facilitated long-term differentiation of hMSCs could be achieved by synergistic effect of dual BMP2 and PDGF in nanofbrous environments.

Abstract LB-028: Mechanically tunable 3D microenvironments modulate tumor cell phenotype: Models of mechanotransduction and drug resistance in osteosarcoma

Abstract The past few decades have seen marked improvements in survival rates of osteosarcoma due to the advent of modern chemotherapy, radiotherapy, and surgical techniques. However, for patients presenting with metastatic disease, five-year survival rates have remained dismal, hovering around 20%. Repeated failures of clinical trials to confirm potential therapeutic options highlight the need for more accurate pre-clinical testing. Currently, the field of cancer research relies heavily on monolayer culture methods on hard plastic or glass in vitro models; there is a dearth of pre-clinical models that accurately recapitulate the tumor-microenvironment interactions. Matrix stiffness has been implicated in modulating intracellular signaling pathways that promote cancer cell survival, proliferation, and stem cell fate. We have developed a novel three-dimensional (3D) tumor model with variable mechanical properties in order to determine the effect of substrate stiffness and tissue architecture on osteosarcoma cell phenotype, plasticity, and response to therapy. We employed coaxial electrospinning techniques to fabricate highly porous fibrous mesh scaffolds that mimic the bone microenvironment. By controlling the ratio of poly(ϵ-caprolactone) (PCL) and gelatin (PCL:gelatin, core:shell, respectively) in constituent fibers, we were able to manipulate the range of tensile moduli of individual fibers over three orders of magnitude, from 68.91 ± 8.77 kPa to 66.05 ± 7.61 MPa. Osteosarcoma cells cultured in these variable mechanical environments responded by modulating the localization and expression of Hippo pathway regulators. YAP downregulation correlated with decreasing fiber stiffness while both YAP and TAZ had decreasing nuclear:cytoplasmic ratio in less stiff environments. Furthermore, the IGF-1/mTOR axis was downregulated in 3D conditions compared to monolayers and a strong upregulation of Sox2, a stem cell transcription factor, was observed in all 3D conditions. Correspondingly, in the presence of agents targeting the IGF-1/mTOR axis, dose response curves to doxorubicin indicated that IC50 values increase with decreasing substrate stiffness. These phenotypic changes indicate that osteosarcoma cells respond to both stiffness and architecture by modulating the Hippo and IGF-1R/mTOR pathways and increasing cancer stem cell qualities and chemoresistance. We sought to validate our model using osteosarcoma patient biopsies. Analysis of tumor samples from 36 osteosarcoma patients confirmed that YAP/TAZ localization and nuclear pIGF-1R/IGF-1R in our 3D models recapitulated phenotypes observed in patient samples. Our models highlight the need for incorporation of mechanical and architectural cues in the preclinical study of cancer biology as these signals have drastic impacts on osteosarcoma phenotypes and responses to therapy. Citation Format: Eric R. Molina, Letitia K. Chim, Maria C. Salazar, Shail M. Mehta, Brian A. Menegaz, Salah-Eddine Cherradi-Lamhamedi, Tejus Satish, David McCall, Sana Mohiuddin, Ana Maria Zaska, Katherine Jane Grande-Allen, Joseph A. Ludwig, Antonios G. Mikos. Mechanically tunable 3D microenvironments modulate tumor cell phenotype: Models of mechanotransduction and drug resistance in osteosarcoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr LB-028.

Microstructured hydrogel scaffolds containing differential density interfaces promote rapid cellular invasion and vascularization

IntroductionInsufficient vascularization of currently available clinical biomaterials has limited their application to optimal wound beds. We designed a hydrogel scaffold with a unique internal microstructure of differential collagen densities to induce cellular invasion and neovascularization. MethodsMicrosphere scaffolds (MSS) were fabricated by encasing 1% (w/v) type 1 collagen microspheres 50–150 μm in diameter in 0.3% collagen bulk. 1% and 0.3% monophase collagen scaffolds and Integra® disks served as controls. Mechanical characterization as well as in vitro and in vivo invasion assays were performed. Cell number and depth of invasion were analyzed using Imaris™. Cell identity was assessed immunohistochemically. ResultsIn vitro, MSS exhibited significantly greater average depth of cellular invasion than Integra® and monophase collagen controls. MSS also demonstrated significantly higher cell counts than controls. In vivo, MSS revealed significantly more cellular invasion spanning the entire scaffold depth at 14 days than Integra®. CD31+ expressing luminal structures suggestive of neovasculature were seen within MSS at 7 days and were more prevalent after 14 days. Multiphoton microscopy of MSS demonstrated erythrocytes within luminal structures after 14 days. ConclusionBy harnessing simple architectural cues to induce cellular migration, MSS holds great potential for clinical translation as the next generation dermal replacement product. Statement of SignificanceLarge skin wounds require tissue engineered dermal substitutes in order to promote healing. Currently available dermal replacement products do not always adequately incorporate into the body, especially in complex wounds, due to poor neovascularization. In this paper, we present a hydrogel with an innovative microarchitecture that is composed of dense type I collagen microspheres suspended in a less-dense collagen bulk. We show that cell invasion into the scaffold is driven solely by mechanical cues inherent within this differential density interface, and that this induces robust vascular cell invasion both in vitro and in a rodent model. Our hydrogel performs favorably compared to the current clinical gold standard, Integra®. We believe this hydrogel scaffold may be the first of the next generation of dermal replacement products.

Stress relaxing hyaluronic acid-collagen hydrogels promote cell spreading, fiber remodeling, and focal adhesion formation in 3D cell culture

The physical and architectural cues of the extracellular matrix (ECM) play a critical role in regulating important cellular functions such as spreading, migration, proliferation, and differentiation. Natural ECM is a complex viscoelastic scaffold composed of various distinct components that are often organized into a fibrillar microstructure. Hydrogels are frequently used as synthetic ECMs for 3D cell culture, but are typically elastic, due to covalent crosslinking, and non-fibrillar. Recent work has revealed the importance of stress relaxation in viscoelastic hydrogels in regulating biological processes such as spreading and differentiation, but these studies all utilize synthetic ECM hydrogels that are non-fibrillar. Key mechanotransduction events, such as focal adhesion formation, have only been observed in fibrillar networks in 3D culture to date. Here we present an interpenetrating network (IPN) hydrogel system based on HA crosslinked with dynamic covalent bonds and collagen I that captures the viscoelasticity and fibrillarity of ECM in tissues. The IPN hydrogels exhibit two distinct processes in stress relaxation, one from collagen and the other from HA crosslinking dynamics. Stress relaxation in the IPN hydrogels can be tuned by modulating HA crosslinker affinity, molecular weight of the HA, or HA concentration. Faster relaxation in the IPN hydrogels promotes cell spreading, fiber remodeling, and focal adhesion (FA) formation – behaviors often inhibited in other hydrogel-based materials in 3D culture. This study presents a new, broadly adaptable materials platform for mimicking key ECM features of viscoelasticity and fibrillarity in hydrogels for 3D cell culture and sheds light on how these mechanical and structural cues regulate cell behavior.

Biomineralization of Recombinant Peptide Scaffolds: Interplay among Chemistry, Architecture, and Mechanics.

Biomineralized scaffolds are an attractive option for bone tissue engineering, being similar to native bone. However, optimization is difficult, due to the complex interplay among architecture, chemistry, and mechanics. Utilizing biomimetic nucleation, linear mineralized scaffolds were created from a collagen type I based recombinant peptide (RCP). Osteoblast mineralization was assessed, in response to changes in scaffold architecture, hydroxyapatite (HA) content, and mechanics. Changes in scaffold pore size (150-450 μm) had little effect on mRNA levels but influenced cell proliferation, achieving a balance between nutrient diffusion and surface area for cell attachment at 300 μm. Increasing the scaffold mechanical strength, from 2.9 to 5.2 kPa, enhanced the expression of osteocalcin, a late marker of mineralization. Further addition of HA, up to 20 wt %, increased osteoblast mineralization, without altering the compressive modulus. Thus, it was shown that architectural cues influence cellular proliferation, while the scaffold chemistry and mechanics independently contribute to gene expression.