Biology subjects

Guldberg, R. E.

Publications and source records attributed to Guldberg, R. E..

13 recordsLinked to original sources

bioRxiv2026

A Multi-Institution Biobanking Pipeline for Primary Human Satellite Cells and Fibro-Adipogenic Progenitors

Satellite Cells (SCs) and Fibro-Adipogenic Progenitors (FAPs) are muscle-resident cell populations crucial for maintaining skeletal muscle homeostasis and coordinating regeneration after injuries. However, primary human SCs and FAPs are difficult to co-isolate, and their broad use in translational research has been limited by a lack of standardized biobanking protocols. Recently, we published a protocol for efficient co-isolation of SCs and FAPs from human skeletal muscle. Here, we extend those efforts to establish a comprehensive pipeline for the cryopreservation, cold-chain transport, and independent-site utilization of human SCs and FAPs. Cells taken through this pipeline maintained lineage-specific markers, including Pax7, MyoD and CD56 for SCs, and PDGFR and TE7 for FAPs, indicating retention of their pre-biobanking phenotype. Furthermore, SCs demonstrate robust myogenic differentiation capacity, and FAPs demonstrate both fibrogenic and adipogenic differentiation capacity post-transport. Finally, previously biobanked SCs were incorporated into in vitro 3D muscle constructs, demonstrating their utility for human-based New Approach Methodologies (NAMs). This framework for multi-site collaboration facilitates broader access to human primary muscle cells, which will improve the scalability and translatability of human-based NAMs for skeletal muscle research.

cell biology↗

bioRxiv2025

Melt Electrowritten Microfiber-Hydrogel Composite Scaffolds for Aligned Muscle Tissue Engineering

Effective regeneration of skeletal muscle with highly aligned fiber architecture remains a significant challenge in tissue engineering. Structural alignment of muscle constructs along with mechanical integrity are crucial for effective engineering of grafts and microphysiological systems. This study engineered a novel composite microfiber-hydrogel platform using melt-electrowriting (MEW) to fabricate high precision microfiber scaffolds from poly({varepsilon}-caprolactone). Three MEW scaffold designs (Isotropic, Aligned T with perpendicular reinforcements, and Aligned X with angled fiber bridges) were developed and fabricated into composite scaffolds with collagen hydrogels and seeded with myoblasts. Aligned X scaffolds with cross-bridge reinforcements exhibited enhanced mechanical strength and continuous alignment without structural interruption that led to highly aligned and multinucleated cellular organization. The incorporation of collagen hydrogel in composite constructs improved cell seeding efficiency, viability, and metabolic activity compared to scaffolds alone. All scaffold designs provided fiber reinforcement that prevented hydrogel contraction over extended culture periods. Critically, the Aligned X composite constructs significantly increased myoblast differentiation and myotube maturation, evidenced by increased myosin heavy chain expression and myotube diameter. Overall, this composite microfiber-hydrogel approach provides a scalable, structurally stable, and highly aligned platform tailored for enhanced muscle tissue engineering applications, representing an advancement towards addressing clinical challenges associated with muscle injuries.

bioengineering↗

bioRxiv2025

Phased affinity-controlled delivery of vascular endothelial growth factor, fibroblast growth factor-2, and platelet derived growth factor enhances in vitro angiogenesis

Angiogenesis, the growth of vasculature from existing blood vessels, requires the coordinated secretion of multiple angiogenic growth factors that each stimulate the cellular recruitment, patterning, and morphogenesis inherent to vascular network formation. Among these secreted factors, vascular endothelial growth factor (VEGF), fibroblast growth factor-2 (FGF-2), and platelet derived growth factor (PDGF) amplify key stages of angiogenesis. Disruptions in their secretion have been implicated in poor vascular network formation. Current methods for exploring variations in the phased presentation of multiple different proteins are limited, which has restricted our ability to explore the effect of growth factor timing on angiogenesis. To address this knowledge gap, we developed affibodies, which are alpha-helical binding proteins, to phase the release of VEGF-165, FGF-2, and PDGF-BB from a single delivery vehicle via specific protein-affibody affinity interactions. We used yeast surface display to engineer three VEGF-, three FGF-2-, and two PDGF-specific affibodies with a wide range of affinities for their target proteins spanning dissociation constants of 3.08 {+/-} 0.21 nM to 4550 {+/-} 590 nM. We demonstrated that the cumulative release of VEGF and FGF-2 is inversely correlated with the strength of the protein-affibody affinity interaction and that hydrogels containing multiple protein-specific affibodies can control the release of VEGF, FGF-2, and PDGF, largely in accordance with the strength of the affinity interactions. Using a rat-derived intact microvascular fragment model of in vitro angiogenesis, we revealed that sequential delivery of soluble VEGF, followed by FGF-2, and then PDGF enhances vascular network length by 2.8-fold and branching by 4.1-fold compared to untreated MVFs. We then designed an affibody-conjugated polyethylene glycol maleimide (PEG-MAL) hydrogel to mimic this sequence of protein delivery, resulting in a 3.0-fold increase in vascular network length and a 2.3-fold increase in vascular branching compared to all other hydrogel compositions and the sequential delivery of soluble growth factors. Changing temporal growth factor presentation with affibody-conjugated hydrogels altered the expression of key angiogenic genes involved in vessel stabilization and destabilization and matrix remodeling. Perivascular coverage measured by the colocalization of lectin and alpha smooth muscle actin staining was similar between all treatment groups, suggesting pericyte recruitment to stabilize expanded vascular networks created by the soluble and affibody-mediated delivery of the optimal sequence of proteins. Overall, this work establishes a new biomaterial platform for modulating the timing of growth factor delivery, enabling the exploration of how temporal variations in protein secretion impact regeneration and development. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=127 SRC="FIGDIR/small/662647v2_ufig1.gif" ALT="Figure 1"> View larger version (41K): org.highwire.dtl.DTLVardef@471657org.highwire.dtl.DTLVardef@10bc491org.highwire.dtl.DTLVardef@1961f9dorg.highwire.dtl.DTLVardef@f5b8ba_HPS_FORMAT_FIGEXP M_FIG C_FIG

bioengineering↗

bioRxiv2023

In vitro development and optimization of cell-laden injectable bioprinted gelatin methacryloyl (GelMA) microgels mineralized on the nanoscale

Bone defects may occur in different sizes and shapes due to trauma, infections, and cancer resection. Autografts are still considered the primary treatment choice for bone regeneration. However, they are hard to source and often create donor-site morbidity. Injectable microgels have attracted much attention in tissue engineering and regenerative medicine due to their ability to replace inert implants with a minimally invasive delivery. Here, we developed novel cell-laden bioprinted gelatin methacrylate (GelMA) injectable microgels, with controllable shapes and sizes that can be controllably mineralized on the nanoscale, while stimulating the response of cells embedded within the matrix. The injectable microgels were mineralized using a calcium and phosphate-rich medium that resulted in nanoscale crystalline hydroxyapatite deposition and increased stiffness within the crosslinked matrix of bioprinted GelMA microparticles. Next, we studied the effect of mineralization in osteocytes, a key bone homeostasis regulator. Viability stains showed that osteocytes were maintained at 98% viability after mineralization with elevated expression of sclerostin in mineralized compared to non-mineralized microgels, indicating that mineralization effectively enhances osteocyte maturation. Based on our findings, bioprinted mineralized GelMA microgels appear to be an efficient material to approximate the bone microarchitecture and composition with desirable control of sample injectability and polymerization. These bone-like bioprinted mineralized biomaterials are exciting platforms for potential minimally invasive translational methods in bone regenerative therapies.

bioengineering↗

bioRxiv2020

Effects of osteogenic ambulatory mechanical stimulation on early stages of BMP-2mediated bone repair

Mechanical loading of bone defects through rehabilitation is a promising therapeutic approach to stimulate repair and reduce the risk of non-union; however, little is known about how therapeutic mechanical stimuli modulate early stages of repair before mineralized bone formation. In a previous study, we established an osteogenic mechanical loading protocol using early ambulatory rehabilitation and a compliant, load-sharing fixator in a rat model of BMP-2 mediated bone defect repair. The objective of this study was to investigate the early effects of osteogenic loading on cytokine expression, tissue composition, and angiogenesis during the first 3 weeks of repair in this model. Using a wireless implantable strain sensor for local measurements of mechanical boundary conditions, finite element simulations showed that osteogenic mechanical loading increased mean compressive strain in defect soft tissue during rehabilitative ambulation at 1 week (load-sharing: -1.54 {+/-} 0.17% vs. load-shielded: -0.76 {+/-} 0.06%), and that strain was amplified in remaining soft tissue regions at 3 weeks as mineralization progressed (load-sharing: -1.89 {+/-} 0.35% vs. load-shielded: -1.38 {+/-} 0.35%). Multivariate analysis of multiplex cytokine arrays revealed that loading significantly altered cytokine expression profiles in the defect tissue at 2 weeks compared to load-shielded defects. Specifically, loading reduced VEGF and increased CXCL5 (LIX) levels. Subsequently, vascular volume in loaded defects was reduced relative to load-shielded defects but similar to intact bone at 3 weeks. Endochondral bone repair was also observed histologically in loaded defects only at 3 weeks. Together, these results demonstrate that moderate ambulatory strains previously shown to stimulate functional bone regeneration significantly alter early angiogenic and cytokine signaling and may promote endochondral ossification in large segmental bone defects. Authors ContributionsB.S.K., N.J.W., and R.E.G. designed the research and performed surgeries; B.S.K., C.E.V., and J.K. performed experiments; B.S.K., C.E.V., J.K., and L.B.W., analyzed data; B.S.K., C.E.V., N.J.W., and R.E.G. wrote the manuscript; All authors interpreted data, critically edited, and have read and approved the final manuscript.

bioengineering↗

bioRxiv2020

Effects of Controlled Dual Growth Factor Delivery on Bone Regeneration Following Composite Bone-Muscle Injury

The objective of this study was to investigate the controlled release of two growth factors (BMP-2 and VEGF) as a treatment strategy for clinically challenging composite injuries, consisting of a segmental bone defect and volumetric muscle loss. This is the first investigation of dual growth factor delivery in a composite injury model using an injectable smart delivery system consisting of heparin microparticles and alginate gel. The loading efficiency of growth factors into these biomaterials was found to be >90%, revealing a strong affinity of VEGF and BMP-2 to heparin and alginate. The system could achieve simultaneous or sequential release of VEGF and BMP-2 by varying the loading strategy. Single growth factor delivery (VEGF or BMP-2 alone) significantly enhanced vascular growth in vitro. However, no synergistic effect was observed for dual growth factor (BMP-2 + VEGF) delivery. Effective bone healing was achieved in all treatment groups (BMP-2, simultaneous or sequential delivery of BMP-2 and VEGF) in the composite injury model. The mechanics of the regenerated bone reached a maximum strength of [~]52% of intact bone with sequential delivery of VEGF and BMP-2. Overall, simultaneous or sequential co-delivery of low-dose BMP-2 and VEGF failed to fully restore the mechanics of bone in this injury model. Given the severity of the composite injury, VEGF alone may not be sufficient to establish mature and stable blood vessels when compared with previous studies co-delivering BMP-2+VEGF enhanced bone tissue regeneration. Hence, future studies are warranted to develop an alternative treatment strategy focusing on better control over growth factor dose, spatiotemporal delivery, and additional growth factors to regenerate fully functional bone tissue. HighlightsO_LIWe developed a smart growth factor delivery system using heparin microparticles and alginate that facilitates tunable delivery of VEGF and BMP-2 in a simultaneous or sequential manner by merely varying the loading strategy. C_LIO_LIIn vitro, both VEGF and BMP-2 alone promoted vascular growth; however, VEGF was significantly more potent, and there was no detectable benefit of co-delivery. C_LIO_LIIn vivo, both BMP-2 alone and co-delivery of VEGF and BMP-2 promoted bone formation in the challenging bone/muscle polytrauma model; however, none of the treatment groups restored biomechanical properties to that of uninjured bone. C_LI

bioengineering↗

bioRxiv2020

3D printing of Microgel-loaded Modular LEGO-like Cages as Instructive Scaffolds for Tissue Engineering

Biomaterial scaffolds have served as the foundation of tissue engineering and regenerative medicine. However, scaffold systems are often difficult to scale in size or shape in order to fit defect-specific dimensions, and thus provide only limited spatiotemporal control of therapeutic delivery and host tissue responses. Here, a lithography-based three-dimensional (3D) printing strategy is used to fabricate a novel miniaturized modular LEGO-like cage scaffold system, which can be assembled and scaled manually with ease. Scalability is based on an intuitive concept of stacking modules, like conventional LEGO blocks, allowing for literally thousands of potential geometric configurations, and without the need for specialized equipment. Moreover, the modular hollow-cage design allows each unit to be loaded with biologic cargo of different compositions, thus enabling controllable and easy patterning of therapeutics within the material in 3D. In summary, the concept of miniaturized cage designs with such straight-forward assembly and scalability, as well as controllable loading properties, is a flexible platform that can be extended to a wide range of materials for improved biological performance. TOC3D printed LEGO-like hollow microcages can be easily assembled, adjoined, and stacked-up to suit the complexity of defect tissues; aid spatial loading of cells and biomolecules; instruct cells migration three-dimensionally; and facilitate cell invasion and neovascularization in-vivo, thus accelerating the process of tissue healing and new tissue formation. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=190 SRC="FIGDIR/small/974204v1_ufig1.gif" ALT="Figure 1"> View larger version (88K): org.highwire.dtl.DTLVardef@1a97a72org.highwire.dtl.DTLVardef@1a60fbeorg.highwire.dtl.DTLVardef@1538f13org.highwire.dtl.DTLVardef@d6345b_HPS_FORMAT_FIGEXP M_FIG C_FIG

bioengineering↗

bioRxiv2020

Mechanical Regulation of Microvascular Angiogenesis

Neovascularization is a critical early step toward successful tissue regeneration during wound healing. While vasculature has long been recognized as highly mechanosensitive (to fluid shear, pulsatile luminal pressure, etc.), the effects of extracellular matrix strains on angiogenesis are poorly understood. Previously, we found that dynamic matrix compression in vivo potently regulated neovascular growth during tissue regeneration; however, whether matrix deformations directly regulate00 angiogenesis remained unknown. Here, we tested the effects of load initiation time, strain magnitude, and mode of compressive deformation (uniform compression vs. compressive indentation that also introduced shear stress) on neovascularization and key angiogenic and mechanotransduction signaling pathways by microvascular fragments in vitro. We hypothesized that neovascularization would be enhanced by delayed, moderate compression and inhibited by early, high magnitude compression and by compressive indentation. Consistent with our hypothesis, early, high magnitude loading inhibited vessel growth, while delayed loading enhanced vessel growth. Compressive indentation led to longer, more branched networks than uniform compression - particularly at high strain magnitude. Gene expression was differentially regulated by time of load initiation; genes associated with active angiogenic sprouts were downregulated by early loading but upregulated by delayed loading. Canonical gene targets of the YAP/TAZ mechanotransduction pathway were increased by loading and abrogated by pharmacological YAP inhibition. Together, these data demonstrate that neovascularization is directly responsive to dynamic matrix strain and is particularly sensitive to the timing of load initiation. This work further identifies putative mechanoregulatory angiogenic mechanisms and implicates a critical role for dynamic mechanical cues in vascularized tissue regeneration. Statement of SignificanceMechanical cues influence tissue regeneration, and although vasculature is known to be mechanically sensitive, remarkably little is known about the effects of bulk extracellular matrix deformation on the nascent vessel networks found in healing tissues. Here, we demonstrated that load initiation time, magnitude, and mode all regulate microvascular growth, as well as upstream angiogenic and mechanotransduction signaling pathways. Across all tested magnitudes and modes, microvascular network formation and upstream signaling were powerfully regulated by the timing of load initiation. This work provides a new foundational understanding of how extracellular matrix mechanics regulate angiogenesis and has critical implications for clinical translation of new regenerative medicine therapies and physical rehabilitation strategies designed to enhance revascularization during tissue regeneration.

bioengineering↗

bioRxiv2020

Development of Systemic Immune Dysregulation in a Rat Trauma Model with Biomaterial-Associated Infection

Orthopedic biomaterial-associated infections remain a large clinical challenge, particularly with open fractures and segmental bone loss. Invasion and colonization of bacteria within immune-privileged canalicular networks of the bone can lead to local, indolent infections that can persist for years without symptoms before eventual catastrophic hardware failure. Host immunity is essential for bacterial clearance and an appropriate healing response, and recent evidence has suggested an association between orthopedic trauma and systemic immune dysregulation and immunosuppression. However, the impact of a local infection on this systemic immune response and subsequent effects on the local response is poorly understood and has not been a major focus for addressing orthopedic injuries and infections. Therefore, this study utilized a model of orthopedic biomaterial-associated infection to investigate the effects of infection on the long-term immune response. Here, despite persistence of a local, indolent infection lacking outward symptoms, there was still evidence of long-term immune dysregulation with systemic increases in MDSCs and decreases in T cells compared to non-infected trauma. Further, the trauma only group exhibited a regulated and coordinated systemic cytokine response, which was not present in the infected trauma group. Locally, the infection group had attenuated macrophage infiltration in the local soft tissue compared to the non-infected group. Our results demonstrate widespread impacts of a localized orthopedic infection on the systemic and local immune responses. Characterization of the immune response to orthopedic biomaterial-associated infection may identify key targets for immunotherapies that could optimize both regenerative and antibiotic interventions, ultimately improving outcomes for these patients.

bioengineering↗

bioRxiv2019

Real-time monitoring of mechanical cues in the regenerative niche reveal dynamic strain magnitudes that enhance bone repair

Mechanical loads exerted on the skeleton during activities such as walking are important regulators of bone repair, but dynamic biomechanical signals are difficult to measure inside the body. The inability to measure the mechanical environment in injured tissues is a significant barrier to developing integrative regenerative and rehabilitative strategies that can accelerate recovery from fracture, segmental bone loss, and spinal fusion. Here we engineered an implantable strain sensor platform and measured strain across a bone defect in real-time throughout rehabilitation. We used the sensor to longitudinally quantify mechanical cues imparted by a load-sharing fixation plate that significantly enhanced bone regeneration in rats. We found that sensor readings correlated with the status of healing, suggesting a potential role for strain sensing as an X-ray-free healing assessment platform. This study demonstrates a promising approach to quantitatively develop and exploit mechanical rehabilitation strategies that enhance bone repair.

bioengineering↗

bioRxiv2019

PGC-1α overexpression partially rescues impaired oxidative and contractile pathophysiology following volumetric muscle loss injury

Volumetric muscle loss (VML) injury is characterized by a non-recoverable loss of muscle fibers due to ablative surgery or severe orthopaedic trauma, that results in chronic functional impairments of the soft tissue. Currently, the effects of VML on the oxidative capacity and adaptability of the remaining injured muscle are unclear. A better understanding of this pathophysiology could significantly shape how VML-injured patients and clinicians approach regenerative medicine and rehabilitation following injury. Herein, the data indicated that VML-injured muscle has diminished mitochondrial content and function (i.e. oxidative capacity), loss of mitochondrial network organization, and attenuated oxidative adaptations to exercise. However, forced PGC-1 over-expression rescued the deficits in oxidative capacity and muscle strength. This implicates physiological activation of PGC1- as a limiting factor in VML-injured muscle adaptive capacity and provides a mechanistic target for regenerative rehabilitation approaches to address the skeletal muscle dysfunction.

physiology↗

bioRxiv2018

Effects of BMP-2 dose and delivery of microvascular fragments on healing of bone defects with concomitant volumetric muscle loss

Traumatic composite bone-muscle injuries, such as open fractures, often require multiple surgical interventions and still typically result in long-term disability. Clinically, a critical indicator of composite injury severity is vascular integrity; vascular damage alone is sufficient to assign an open fracture to the most severe category. Challenging bone injuries are often treated with bone morphogenetic protein 2 (BMP-2), an osteoinductive growth factor, delivered on collagen sponge. Previous studies in a composite defect model found that a minimally bridging dose in the segmental defect model was unable to overcome concomitant muscle damage, but the effect of BMP dose on composite injuries has not yet been studied. Here, we test the hypotheses that BMP-2-mediated functional regeneration of composite extremity injuries is dose dependent and can be further enhanced via co-delivery of adipose-derived microvascular fragments (MVF), which have been previously shown to increase tissue vascular volume. Although MVF treatment did not improve healing outcomes, we observed a significant BMP-2 dose-dependent increase in regenerated bone volume and biomechanical properties. While high dose BMP-2 delivery can induce heterotopic ossification (HO) and increased inflammation, the maximum 10 g dose used in this study did not result in HO and was associated with a lower circulating inflammatory cytokine profile than the low dose (2.5 g) group. These data support the potential benefits of an increased, though still moderate, BMP-2 dose for treatment of bone defects with concomitant muscle damage. Future work to improve vascularization may further improve healing.

bioengineering↗

bioRxiv2017

Wireless Implantable Sensor For Non-Invasive, Longitudinal Quantification Of Axial Strain Across Rodent Long Bone Defects

Bone development, maintenance, and regeneration are remarkably sensitive to mechanical cues. Consequently, mechanical stimulation has long been sought as a putative target to promote endogenous healing after fracture. Given the transient nature of bone repair, tissue-level mechanical cues evolve rapidly over time after injury and are challenging to measure non-invasively. The objective of this work was to develop and characterize an implantable strain sensor for non-invasive monitoring of axial strain across a rodent femoral defect during functional activity. Herein, we present the design, characterization, and in vivo demonstration of the devices capabilities for quantitatively interrogating physiological dynamic strains during bone regeneration. Ex vivo experimental characterization of the device showed that it exceeded the technical requirements for sensitivity, signal resolution, and electromechanical stability. The digital telemetry minimized power consumption, enabling long-term intermittent data collection. Devices were implanted in a rat 6 mm femoral segmental defect model and after three days, data were acquired wirelessly during ambulation and synchronized to corresponding radiographic videos, validating the ability of the sensor to non-invasively measure strain in real-time. Lastly, in vivo strain measurements were utilized in a finite element model to estimate the strain distribution within the defect region. Together, these data indicate the sensor is a promising technology to quantify local tissue mechanics in a specimen specific manner, facilitating more detailed investigations into the role of the mechanical environment in dynamic skeletal healing and remodeling.

bioengineering↗