Gold nanoshell-localized photothermal ablation of prostate tumors in a clinical pilot device study.

Biocompatible gold nanoparticles designed to absorb light at wavelengths of high tissue transparency have been of particular interest for biomedical applications. The ability of such nanoparticles to convert absorbed near-infrared light to heat and induce highly localized hyperthermia has been shown to be highly effective for photothermal cancer therapy, resulting in cell death and tumor remission in a multitude of preclinical animal models. Here we report the initial results of a clinical trial in which laser-excited gold-silica nanoshells (GSNs) were used in combination with magnetic resonance-ultrasound fusion imaging to focally ablate low-intermediate-grade tumors within the prostate. The overall goal is to provide highly localized regional control of prostate cancer that also results in greatly reduced patient morbidity and improved functional outcomes. This pilot device study reports feasibility and safety data from 16 cases of patients diagnosed with low- or intermediate-risk localized prostate cancer. After GSN infusion and high-precision laser ablation, patients underwent multiparametric MRI of the prostate at 48 to 72 h, followed by postprocedure mpMRI/ultrasound targeted fusion biopsies at 3 and 12 mo, as well as a standard 12-core systematic biopsy at 12 mo. GSN-mediated focal laser ablation was successfully achieved in 94% (15/16) of patients, with no significant difference in International Prostate Symptom Score or Sexual Health Inventory for Men observed after treatment. This treatment protocol appears to be feasible and safe in men with low- or intermediate-risk localized prostate cancer without serious complications or deleterious changes in genitourinary function.

Synthetic ECM: Bioactive Synthetic Hydrogels for 3D Tissue Engineering.

The specific microenvironment that cells reside in fundamentally impacts their broader function in tissues and organs. At its core, this microenvironment is composed of precise arrangements of cells that encourage homotypic and heterotypic cell-cell interactions, biochemical signaling through soluble factors like cytokines, hormones, and autocrine, endocrine, or paracrine secretions, and the local extracellular matrix (ECM) that provides physical support and mechanobiological stimuli, and further regulates biochemical signaling through cell-ECM interactions like adhesions and growth factor sequestering. Each cue provided in the microenvironment dictates cellular behavior and, thus, overall potential to perform tissue and organ specific function. It follows that in order to recapitulate physiological cell responses and develop constructs capable of replacing damaged tissue, we must engineer the cellular microenvironment very carefully. Many great strides have been made toward this goal using various three-dimensional (3D) tissue culture scaffolds and specific media conditions. Among the various 3D biomimetic scaffolds, synthetic hydrogels have emerged as a highly tunable and tissue-like biomaterial well-suited for implantable tissue-engineered constructs. Because many synthetic hydrogel materials are inherently bioinert, they minimize unintentional cell responses and thus are good candidates for long-term implantable grafts, patches, and organs. This review will provide an overview of commonly used biomaterials for forming synthetic hydrogels for tissue engineering applications and techniques for modifying them to with bioactive properties to elicit the desired cell responses.

Chemically Orthogonal Protein Ligation Domains for Independent Control of Hydrogel Modification with Adhesive Ligands and Growth Factors.

The twin, chemically orthogonal protein ligation domains, SpyCatcher and SnoopCatcher, were used to link two engineered proteins into poly(ethylene glycol) (PEG) hydrogels in order to control both endothelial cell adhesion and material-mediated pro-mitotic stimulation. SpyCatcher was appended with an N-terminal adhesion ligand RGDS to form RGDS-SC, and SnoopCatcher was appended with the vascular endothelial growth factor (VEGF)-mimetic peptide QK to form QK-SnpC. QK-SnpC formed a spontaneous covalent bond with SnoopTag peptide with 40% reaction efficiency, both in solution, in a PEG gel containing SnoopTag peptide, and in a PEG gel with both SnoopTag and SpyTag sites. QK-SnpC added to cell culture media enhanced endothelial cell proliferation compared to a negative control, and was statistically indistinguishable from the positive control of 130 pM VEGF165. Endothelial cells seeded onto PEG gels presenting both RGDS-SC and QK-SnpC showed ∼50% of cells actively proliferating (defined as Ki67+), compared to ∼31% of cells seeded on gels presenting RGDS-SC alone. These results show that complementary nondiffusing biochemical signals can be linked into PEG-DA hydrogels simultaneously using 'Catcher-based ligation strategies, thereby inducing more nuanced cell-material interactions.

Cell-Compatible, Site-Specific Covalent Modification of Hydrogel Scaffolds Enables User-Defined Control over Cell-Material Interactions.

SpyCatcher, a 15 kDa protein domain that spontaneously forms a site-specific covalent bond with the 13 amino acid peptide SpyTag, was used to covalently link a model recombinant protein containing a SpyCatcher domain and the adhesive ligand Arg-Gly-Asp-Ser (RGDS) (RGDS-SC) into SpyTag-containing poly(ethylene glycol) (PEG) hydrogels. This new strategy for covalent immobilization of proteins or peptides provides an easy and gentle mechanism for biochemical modification of hydrogels. Labeling efficiency was approximately 100% when soluble RGDS-SC was applied to SpyTag-containing hydrogels at a 1:1 molar ratio. RGDS-SC remained stably bound throughout the 5 days of rinsing, and 3T3 fibroblasts were able to adhere to PEG gels presenting RGDS-SC, but did not adhere when the scrambled amino acid sequence RDGS was presented instead. Fibroblasts encapsulated within 3D cell-degradable PEG hydrogels containing SpyTag did not spread until RGDS-SC was added to the gels, at which point cell spreading was induced. This cell-friendly site-specific ligation strategy could have great utility in driving specific cellular outcomes using biochemically dynamic hydrogels.

Dynamic Ligand Presentation in Biomaterials.

The native cell microenvironment is extraordinarily dynamic, with reciprocal regulation pathways between cells and the extracellular matrix guiding many physiological processes, such as cell migration, stem cell differentiation, and tissue formation. Providing the correct sequence of biochemical cues to cells, both in vivo and in vitro, is critical for triggering specific biological outcomes. There has been a diversity of methods developed for exposing cells in culture to spatiotemporally varying cues, many of which have centered on dynamic control over cell-material interactions in an attempt to recapitulate the role of the extracellular matrix in cell signaling. This review highlights several mechanisms that have been employed to control bioactive ligand presentation in biomaterials, and looks ahead toward the potential for genetically encoded approaches to dynamically regulate material bioactivity using light.

Bioactive poly(ethylene glycol) hydrogels to recapitulate the HSC niche and facilitate HSC expansion in culture.

Hematopoietic stem cells (HSCs) have been used therapeutically for decades, yet their widespread clinical use is hampered by the inability to expand HSCs successfully in vitro. In culture, HSCs rapidly differentiate and lose their ability to self-renew. We hypothesize that by mimicking aspects of the bone marrow microenvironment in vitro we can better control the expansion and differentiation of these cells. In this work, derivatives of poly(ethylene glycol) diacrylate hydrogels were used as a culture substrate for hematopoietic stem and progenitor cell (HSPC) populations. Key HSC cytokines, stem cell factor (SCF) and interferon-γ (IFNγ), as well as the cell adhesion ligands RGDS and connecting segment 1 were covalently immobilized onto the surface of the hydrogels. With the use of SCF and IFNγ, we observed significant expansion of HSPCs, ∼97 and ∼104 fold respectively, while maintaining c-kit(+) lin(-) and c-kit(+) Sca1(+) lin(-) (KSL) populations and the ability to form multilineage colonies after 14 days. HSPCs were also encapsulated within degradable poly(ethylene glycol) hydrogels for three-dimensional culture. After expansion in hydrogels, ∼60% of cells were c-kit(+), demonstrating no loss in the proportion of these cells over the 14 day culture period, and ∼50% of colonies formed were multilineage, indicating that the cells retained their differentiation potential. The ability to tailor and use this system to support HSC growth could have implications on the future use of HSCs and other blood cell types in a clinical setting.

Hyaluronan Hydrogels for a Biomimetic Spongiosa Layer of Tissue Engineered Heart Valve Scaffolds.

Advanced tissue engineered heart valves must be constructed from multiple materials to better mimic the heterogeneity found in the native valve. The trilayered structure of aortic valves provides the ability to open and close consistently over a full human lifetime, with each layer performing specific mechanical functions. The middle spongiosa layer consists primarily of proteoglycans and glycosaminoglycans, providing lubrication and dampening functions as the valve leaflet flexes open and closed. In this study, hyaluronan hydrogels were tuned to perform the mechanical functions of the spongiosa layer, provide a biomimetic scaffold in which valve cells were encapsulated in 3D for tissue engineering applications, and gain insight into how valve cells maintain hyaluronan homeostasis within heart valves. Expression of the HAS1 isoform of hyaluronan synthase was significantly higher in hyaluronan hydrogels compared to blank-slate poly(ethylene glycol) diacrylate (PEGDA) hydrogels. Hyaluronidase and matrix metalloproteinase enzyme activity was similar between hyaluronan and PEGDA hydrogels, even though these scaffold materials were each specifically susceptible to degradation by different enzyme types. KIAA1199 was expressed by valve cells and may play a role in the regulation of hyaluronan in heart valves. Cross-linked hyaluronan hydrogels maintained healthy phenotype of valve cells in 3D culture and were tuned to approximate the mechanical properties of the valve spongiosa layer. Therefore, hyaluronan can be used as an appropriate material for the spongiosa layer of a proposed laminate tissue engineered heart valve scaffold.

3D biofabrication strategies for tissue engineering and regenerative medicine.

Over the past several decades, there has been an ever-increasing demand for organ transplants. However, there is a severe shortage of donor organs, and as a result of the increasing demand, the gap between supply and demand continues to widen. A potential solution to this problem is to grow or fabricate organs using biomaterial scaffolds and a person's own cells. Although the realization of this solution has been limited, the development of new biofabrication approaches has made it more realistic. This review provides an overview of natural and synthetic biomaterials that have been used for organ/tissue development. It then discusses past and current biofabrication techniques, with a brief explanation of the state of the art. Finally, the review highlights the need for combining vascularization strategies with current biofabrication techniques. Given the multitude of applications of biofabrication technologies, from organ/tissue development to drug discovery/screening to development of complex in vitro models of human diseases, these manufacturing technologies can have a significant impact on the future of medicine and health care.

Gadolinium-conjugated gold nanoshells for multimodal diagnostic imaging and photothermal cancer therapy.

Multimodal imaging offers the potential to improve diagnosis and enhance the specificity of photothermal cancer therapy. Toward this goal, gadolinium-conjugated gold nanoshells are engineered and it is demonstrated that they enhance contrast for magnetic resonance imaging, X-ray, optical coherence tomography, reflectance confocal microscopy, and two-photon luminescence. Additionally, these particles effectively convert near-infrared light to heat, which can be used to ablate cancer cells. Ultimately, these studies demonstrate the potential of gadolinium-nanoshells for image-guided photothermal ablation.

Neurogenic Cell Behavior in 3D Culture Enhanced Within a Highly Compliant Synthetic Hydrogel Platform Formed via Competitive Crosslinking.

Purpose: Scaffold materials that better support neurogenesis are still needed to improve cell therapy outcomes for neural tissue damage. We have used a modularly tunable, highly compliant, degradable hydrogel to explore the impacts of hydrogel compliance stiffness on neural differentiation. Here we implemented competitive matrix crosslinking mechanics to finely tune synthetic hydrogel moduli within soft tissue stiffnesses, a range much softer than typically achievable in synthetic crosslinked hydrogels, providing a modularly controlled and ultrasoft 3D culture model which supports and enhances neurogenic cell behavior. Methods: Soluble competitive allyl monomers were mixed with proteolytically-degradable poly(ethylene glycol) diacrylate derivatives and crosslinked to form a matrix, and resultant hydrogel stiffness and diffusive properties were evaluated. Neural PC12 cells or primary rat fetal neural stem cells (NSCs) were encapsulated within the hydrogels, and cell morphology and phenotype were investigated to understand cell-matrix interactions and the effects of environmental stiffness on neural cell behavior within this model. Results: Addition of allyl monomers caused a concentration-dependent decrease in hydrogel compressive modulus from 4.40 kPa to 0.26 kPa (natural neural tissue stiffness) without influencing soluble protein diffusion kinetics through the gel matrix. PC12 cells encapsulated in the softest hydrogels showed significantly enhanced neurite extension in comparison to PC12s in all other hydrogel stiffnesses tested. Encapsulated neural stem cells demonstrated significantly greater spreading and elongation in 0.26 kPa alloc hydrogels than in 4.4 kPa hydrogels. When soluble growth factor deprivation (for promotion of neural differentiation) was evaluated within the neural stiffness gels (0.26 kPa), NSCs showed increased neuronal marker expression, indicating early enhancement of neurogenic differentiation. Conclusions: Implementing allyl-acrylate crosslinking competition reduced synthetic hydrogel stiffness to provide a supportive environment for 3D neural tissue culture, resulting in enhanced neurogenic behavior of encapsulated cells. These results indicate the potential suitability of this ultrasoft hydrogel system as a model platform for further investigating environmental factors on neural cell behavior. Supplementary Information: The online version contains supplementary material available at 10.1007/s12195-024-00794-2.

Detection of fluorescent protein mechanical switching in cellulo.

The ability of cells to sense and respond to mechanical forces is critical in many physiological and pathological processes. However, determining the mechanisms by which forces affect protein function inside cells remains challenging. Motivated by in vitro demonstrations of fluorescent proteins (FPs) undergoing reversible mechanical switching of fluorescence, we investigated whether force-sensitive changes in FP function could be visualized in cells. Guided by a computational model of FP mechanical switching, we develop a formalism for its detection in Förster resonance energy transfer (FRET)-based biosensors and demonstrate its occurrence in cellulo within a synthetic actin crosslinker and the mechanical linker protein vinculin. We find that in cellulo mechanical switching is reversible and altered by manipulation of cell force generation, external stiffness, and force-sensitive bond dynamics of the biosensor. This work describes a framework for assessing FP mechanical stability and provides a means of probing force-sensitive protein function inside cells.

Detection of Fluorescent Protein Mechanical Switching in Cellulo.

The ability of cells to sense and respond to mechanical forces is critical in many physiological and pathological processes. However, the mechanisms by which forces affect protein function inside cells remain unclear. Motivated by in vitro demonstrations of fluorescent proteins (FPs) undergoing reversible mechanical switching of fluorescence, we investigated if force-sensitive changes in FP function could be visualized in cells. Guided by a computational model of FP mechanical switching, we develop a formalism for its detection in Förster resonance energy transfer (FRET)-based biosensors and demonstrate its occurrence in cellulo in a synthetic actin-crosslinker and the mechanical linker protein vinculin. We find that in cellulo mechanical switching is reversible and altered by manipulation of cellular force generation as well as force-sensitive bond dynamics of the biosensor. Together, this work describes a new framework for assessing FP mechanical stability and provides a means of probing force-sensitive protein function inside cells. MOTIVATION: The ability of cells to sense mechanical forces is critical in developmental, physiological, and pathological processes. Cells sense mechanical cues via force-induced alterations in protein structure and function, but elucidation of the molecular mechanisms is hindered by the lack of approaches to directly probe the effect of forces on protein structure and function inside cells. Motivated by in vitro observations of reversible fluorescent protein mechanical switching, we developed an approach for detecting fluorescent protein mechanical switching in cellulo . This enables the visualization of force-sensitive protein function inside living cells.

Nitric oxide-releasing polymeric microspheres improve diabetes-related erectile dysfunction.

INTRODUCTION: We have used a long-acting nitric oxide (NO)-releasing polymer to develop injectable biodegradable microspheres capable of localized NO release over prolonged periods of time. AIM: The aim of this study was to evaluate the therapeutic potential of these microspheres for diabetes-related erectile dysfunction (ED) in the rat model. METHODS: NO-releasing microspheres were incubated in physiologic buffer, and in vitro NO release was measured using a Griess assay. To ensure no migration, microspheres were fluorescently tagged and injected into the corpus cavernosum of adult rats, and fluorescent imaging was performed weekly for 4 weeks, at which point rats were sacrificed. To assess physiologic efficacy, diabetes was induced in 40 rats using streptozotocin (STZ), whereas 10 rats were kept as age-matched controls. Diabetic rats were divided into four groups: no treatment, sildenafil, NO-releasing microspheres, and combination therapy. For each rat, the cavernosal nerve (CN) was stimulated at various voltages, and intracavernosal pressure (ICP) and mean arterial pressure (MAP) were measured via corpus cavernosum and carotid artery catheterization, respectively. Long-term efficacy was determined by injecting diabetic rats with microspheres and measuring erectile response at predetermined intervals for up to 5 weeks. MAIN OUTCOME MEASURES: Erectile response was determined via calculation of mean peak ICP/MAP and area under curve (AUC) for each experimental group. RESULTS: Under physiologic conditions in vitro, microspheres continued NO release for up to 4 weeks. Fluorescent imaging revealed no detectable signal in tissues besides cavernosal tissue at 4 weeks postinjection. Upon CN stimulation, peak ICP/MAP ratio and AUC of diabetic rats improved significantly (P < 0.05) in microsphere and combination therapy groups compared with no treatment and sildenafil groups. In long-term efficacy studies, microspheres augmented the effect of sildenafil for 3 weeks following injection (P < 0.05). CONCLUSIONS: NO-releasing microspheres significantly improved erectile response in diabetic rats for 3 weeks and hence offer a promising approach to ED therapy, either as monotherapy or combination therapy.

Antibiotic exposure is associated with decreased risk of psychiatric disorders.

Objective: This study sought to investigate the relationship between antibiotic exposure and subsequent risk of psychiatric disorders. Methods: This retrospective cohort study used a national database of 69 million patients from 54 large healthcare organizations. We identified a cohort of 20,214 (42.5% male; 57.9 ± 15.1 years old [mean ± SD]) adults without prior neuropsychiatric diagnoses who received antibiotics during hospitalization. Matched controls included 41,555 (39.6% male; 57.3 ± 15.5 years old) hospitalized adults without antibiotic exposure. The two cohorts were balanced for potential confounders, including demographics and variables with potential to affect: the microbiome, mental health, medical comorbidity, and overall health status. Data were stratified by age and by sex, and outcome measures were assessed starting 6 months after hospital discharge. Results: Antibiotic exposure was consistently associated with a significant decrease in the risk of novel mood disorders and anxiety and stressor-related disorders in: men (mood (OR 0.84, 95% CI 0.77, 0.91), anxiety (OR 0.88, 95% CI 0.82, 0.95), women (mood (OR 0.94, 95% CI 0.89,1.00), anxiety (OR 0.93, 95% CI 0.88, 0.98), those who are 26-49 years old (mood (OR 0.87, 95% CI 0.80, 0.94), anxiety (OR 0.90, 95% CI 0.84, 0.97)), and in those ≥50 years old (mood (OR 0.91, 95% CI 0.86, 0.97), anxiety (OR 0.92, 95% CI 0.87, 0.97). Risk of intentional harm and suicidality was decreased in men (OR 0.73, 95% CI 0.55, 0.98) and in those ≥50 years old (OR 0.67, 95% CI 0.49, 0.92). Risk of psychotic disorders was also decreased in subjects ≥50 years old (OR 0.83, 95 CI: 0.69, 0.99). Conclusion: Use of antibiotics in the inpatient setting is associated with protective effects against multiple psychiatric outcomes in an age- and sex-dependent manner.

Integration of Self-Assembled Microvascular Networks with Microfabricated PEG-Based Hydrogels.

Despite tremendous efforts, tissue engineered constructs are restricted to thin, simple tissues sustained only by diffusion. The most significant barrier in tissue engineering is insufficient vascularization to deliver nutrients and metabolites during development in vitro and to facilitate rapid vascular integration in vivo. Tissue engineered constructs can be greatly improved by developing perfusable microvascular networks in vitro in order to provide transport that mimics native vascular organization and function. Here a microfluidic hydrogel is integrated with a self-assembling pro-vasculogenic co-culture in a strategy to perfuse microvascular networks in vitro. This approach allows for control over microvascular network self-assembly and employs an anastomotic interface for integration of self-assembled micro-vascular networks with fabricated microchannels. As a result, transport within the system shifts from simple diffusion to vessel supported convective transport and extra-vessel diffusion, thus improving overall mass transport properties. This work impacts the development of perfusable prevascularized tissues in vitro and ultimately tissue engineering applications in vivo.

A bioresponsive hydrogel tuned to chondrogenesis of human mesenchymal stem cells.

Cartilage tissue engineering aims to replace damaged or diseased tissue with a functional regenerate that restores joint function. Scaffolds are used to deliver cells and facilitate tissue development, but they can also interfere with the structural assembly of the cartilage matrix. Biodegradable scaffolds have been proposed as a means to improve matrix deposition and the biomechanical properties of neocartilage. The challenge is designing scaffolds with appropriate degradation rates, ideally such that scaffold degradation is proportional to matrix deposition. In this study, we developed a bioresponsive hydrogel with cell-mediated degradation aligned to the chondrogenic differentiation of human mesenchymal stem cells (hMSCs). We identified matrix metalloproteinase 7 (MMP7) as an enzyme with a temporal expression pattern that corresponded with cartilage development. By embedding MMP7 peptide substrates within a poly(ethylene glycol) diacrylate backbone, we built MMP7-sensitive hydrogels with distinct degradation rates. When MMP7-sensitive scaffolds were compared with nondegradable scaffolds in vitro, photoencapsulated hMSCs produced neocartilage constructs with more extensive collagenous matrices, as demonstrated through immunohistochemistry and biochemical quantification of matrix molecules. Furthermore, these changes translated into an increased dynamic compressive modulus. This work presents a practical strategy for designing biomaterials uniquely tuned to individual biological processes.

Reductionist Three-Dimensional Tumor Microenvironment Models in Synthetic Hydrogels.

The tumor microenvironment (TME) plays a determining role in everything from disease progression to drug resistance. As such, in vitro models which can recapitulate the cell-cell and cell-matrix interactions that occur in situ are key to the investigation of tumor behavior and selecting effective therapeutic drugs. While naturally derived matrices can retain the dimensionality of the native TME, they lack tunability and batch-to-batch consistency. As such, many synthetic polymer systems have been employed to create physiologically relevant TME cultures. In this review, we discussed the common semi-synthetic and synthetic polymers used as hydrogel matrices for tumor models. We reviewed studies in synthetic hydrogels which investigated tumor cell interactions with vasculature and immune cells. Finally, we reviewed the utility of these models as chemotherapeutic drug-screening platforms, as well as the future directions of the field.

Adding Dynamic Biomolecule Signaling to Hydrogel Systems via Tethered Photolabile Cell-Adhesive Proteins.

Sequential biochemical signaling events direct key native tissue processes including disease progression, wound healing and angiogenesis, and tissue regeneration. While in vitro modeling of these processes is critical to understanding endogenous tissue behavior and improving therapeutic outcomes, current models inadequately recapitulate the dynamism of these signaling events. Even the most advanced current synthetic tissue culture constructs are restricted in their capability to sequentially add and remove the same molecule to model transient signaling. Here, we developed a genetically encoded method for reversible biochemical signaling within poly(ethylene glycol) (PEG)-based hydrogels for greater accuracy of modeling tissue regeneration within a reductionist environment. We designed and implemented a recombinant protein with a SpyCatcher domain connected to a cell-adhesive RGDS peptide domain by a light-cleavable domain known as PhoCl. This protein was shown to bind to SpyTag-functionalized PEG-matrices via SpyTag-SpyCatcher isopeptide bonding to present RGDS adhesive ligands to cells. Upon 405 nm light exposure, the PhoCl domain was cleaved to subsequently release the RGDS peptide, which diffused out of the matrix. This system was implemented to confer reversible adhesion of 3T3 fibroblasts to the PEG-based hydrogel surface in 2D culture (73.36 ± 21.47% cell removal upon cell-compatible light exposure) and temporal control over cell spreading over time in 3D culture within cell-degradable PEG-based hydrogels, demonstrating the capability of this system to present dynamic signaling events to cells toward modeling native tissue processes within in a controlled, ECM-mimetic matrix.

Tunable PEG Hydrogels for Discerning Differential Tumor Cell Response to Biomechanical Cues.

Increased extracellular matrix (ECM) density in the tumor microenvironment has been shown to influence aspects of tumor progression such as proliferation and invasion. Increased matrix density means cells experience not only increased mechanical properties, but also a higher density of bioactive sites. Traditional in vitro ECM models like Matrigel and collagen do not allow these properties to be investigated independently. In this work, a poly(ethylene glycol)-based scaffold is used which modifies with integrin-binding sites for cell attachment and matrix metalloproteinase 2 and 9 sensitive sites for enzyme-mediated degradation. The polymer backbone density and binding site concentration are independently tuned and the effect each of these properties and their interaction have on the proliferation, invasion, and focal complex formation of two different tumor cell lines is evaluated. It is seen that the cell line of epithelial origin (Hs 578T, triple negative breast cancer) proliferates more, invades less, and forms more mature focal complexes in response to an increase in matrix adhesion sites. Conversely, the cell line of mesenchymal origin (HT1080, fibrosarcoma) proliferates more in 2D culture but less in 3D culture, invades less, and forms more mature focal complexes in response to an increase in matrix stiffness.

Cell-based gene therapy for repair of critical size defects in the rat fibula.

More than a decade has passed since the first experiments using adenovirus-transduced cells expressing bone morphogenetic protein 2 were performed for the synthesis of bone. Since this time, the field of bone gene therapy has tackled many issues surrounding safety and efficacy of this type of strategy. We present studies examining the parameters of the timing of bone healing, and remodeling when heterotopic ossification (HO) is used for bone fracture repair using an adenovirus gene therapy approach. We use a rat fibula defect, which surprisingly does not heal even when a simple fracture is introduced. In this model, the bone quickly resorbs most likely due to the non-weight bearing nature of this bone in rodents. Using our gene therapy system robust HO can be introduced at the targeted location of the defect resulting in bone repair. The HO and resultant bone healing appeared to be dose dependent, based on the number of AdBMP2-transduced cells delivered. Interestingly, the HO undergoes substantial remodeling, and assumes the size and shape of the missing segment of bone. However, in some instances we observed some additional bone associated with the repair, signifying that perhaps the forces on the newly forming bone are inadequate to dictate shape. In all cases, the HO appeared to fuse into the adjacent long bone. The data collectively indicates that the use of BMP2 gene therapy strategies may vary depending on the location and nature of the defect. Therefore, additional parameters should be considered when implementing such strategies.