Abstract The outer blood-retinal barrier (OBRB) is the primary interface through which systemically circulating drugs reach the retina. A tool that measures delivery across this barrier would support the development of targeted therapies as alternatives to repeated intravitreal injection, and the screening of drugs that reach the retina as an off-target toxicity. Such a tool should deliver drugs fluidically through a vascular compartment, measure transport across the retinal pigment epithelium (RPE), and display disease phenotypes relevant to efficacy. Here we adapt the µSiM platform, which places epithelium and endothelium in direct juxtaposition across a permeable, optically transparent silicon nitride nanomembrane. ARPE-19 and human umbilical vein endothelial cells (HUVECs) were used as development cell sources. ARPE-19 monocultures reached a transepithelial electrical resistance of 68 ± 26 Ω cm 2 by 28 days, and ARPE-19 + HUVEC co-cultures reached a small-molecule permeability of 6.34 ± 1.3 × 10 −4 cm min −1 within 14 days, a state reported elsewhere only after longer culture. The barriers developed an intervening basement membrane. Drugs perfused through the basal vascular channel crossed into an open apical well, where sampling and mass spectrometry showed transport correlating with lipophilicity, as reported in vivo. The device also displayed two clinically relevant phenotypes. Digoxin at a clinically toxic concentration reduced viability in the co-barrier by about half and doubled permeability. In a vascularized configuration, VEGF drove endothelial invasion of the RPE layer, as seen in neovascular AMD. The µSiM-OBRB therefore satisfies basic design criteria for measurement of drug bioavailability, toxicity, and efficacy.

Decellularized bone allografts are used in approximately 1/3 of grafting procedures and are preferred in treating critical-size bone defects, as volumetric constraints limit autografts. However, allografts demonstrate high failure rates, with 60 % of allografts failing within 10-years post-implantation. Allograft failure is linked to poor graft integration, which directly results from lack of periosteum, which surrounds bone and is necessary for successful bone healing. Therefore, a tissue-engineered periosteum (TEP) is a promising approach to recapitulate the missing periosteum and promote allograft healing. We have systematically developed an enzymatically degradable poly(ethylene glycol) (PEG) hydrogel with encapsulated mouse mesenchymal stem cells and osteoprogenitor cells, recapitulating key periosteal paracrine signals and producing improvements in bone allograft healing. While successful TEP-mediated allograft healing has been observed, previous studies have been limited to short-term healing (up to 16-weeks), which has yet to enable the observation of TEP-modified allograft healing resolution. To this end, this study extended evaluation of allograft healing in a murine femur defect model up to 12-months post-implantation. TEP-modified allografts demonstrated improvements in key bone healing outcomes, including graft vascularization and bone callus formation, at early time points (up to 9-weeks post-implantation), but improvements in healing outcomes compared to unmodified allografts were lost after 4-months post-implantation. In addition, unmodified allografts displayed incomplete healing up to 12-months post-implantation, with significant fibrotic tissue, incomplete graft remodeling, and inferior biomechanical strength observed. Given these results, future TEP designs should support long-term healing and graft remodeling to promote resolution of TEP-mediated graft healing in a clinically relevant timeline.

Thiol-Michael addition reactions are widely used for forming cytocompatible and well-defined hydrogels. Numerous types of Michael acceptors have been implemented in these reactions; while maleimides enable rapid crosslinking under physiological conditions and are commonly used for their simplicity, slower-reacting electrophiles such as vinyl sulfones and acrylates offer distinct advantages including improved network homogeneity and ease of handling because of the slower reaction rates. Additionally, thiol-acrylate adducts are hydrolytically labile, whereas thiol-vinyl sulfone adducts are comparably more stable in aqueous environments. Building on our previous work demonstrating radical-mediated degradation of thiol-maleimide hydrogels, we sought to determine whether other thiol-Michael adducts are similarly susceptible to cleavage by radical species. Using both linear and network-forming polymer systems, we found that both Michael-adduct types undergo radical-mediated degradation to varying extents. Furthermore, acrylates are far more prone to radical homopolymerization, enabling semi-orthogonal degradation modes in hydrogels, wherein hydrolytic and radical responses are independently programmed according to the chemical structure and stoichiometric excess of the Michael acceptor. Extending the results of these findings in networks synthesized via thiol-Michael addition, we also observed similar radical-mediated degradation behavior in thiol-norbornene networks formed via thiol-ene photopolymerization, suggesting that even electron-rich thioethers are degradable under sufficiently aggressive initiation conditions where the concentration of radicals exceeds that of the crosslinks. Together, these results extend the chemical space for engineering hydrogels with variable degradation profiles and illustrate design principles for tuning material responses to multiple chemical stimuli.

A common strategy for promoting bone allograft healing is the design of tissue-engineered periosteum (TEP) to orchestrate host-tissue infiltration. However, evaluating requires costly and time-consuming in vivo studies. Therefore, in vitro assays are necessary to expedite TEP designs. Since angiogenesis is a critical process orchestrated by the periosteum, this study investigates in vitro 3D cell spheroid vascularization as a predictive tool for TEP-mediated in vivo healing. Spheroids of human umbilical vein endothelial cells (HUVECs) and human mesenchymal stem cells (hMSCs) are encapsulated in enzymatically-degradable poly (ethylene glycol)-based hydrogels and sprout formation, network formation, and angiogenic growth factor secretion are quantified. Hydrogels are also evaluated as TEP-modified allografts for in vivo bone healing with graft vascularization, callus formation, and biomechanical strength quantified as healing metrics. Evaluation of hydrogels highlights the importance of degradation, with 24-fold greater day 1 sprouts observed in degradable hydrogels in vitro and 4-fold greater graft-localized vascular volume at 6-weeks in vivo compared to non-degradable hydrogels. Correlations between in vitro and in vivo studies elucidate linear relationships when comparing in vitro sprout formation and angiocrine production with 3- and 6-week in vivo graft vascularization, 3-week cartilage callus, and 6-week bone callus, with a Pearson's R2 value equal to 0.97 for the linear correlation between in vitro sprout formation and 6-week in vivo vascular volume. Non-linear relationships are found between in vitro measures and bone torque strength at week 6. These correlations suggest that the in vitro sprouting assay has predictive power for in vivo vascularization and bone allograft healing.

Although allografts remain the gold standard for treating critical-size bone defects, ~60% fail within 10 years of implantation. To emulate periosteum-mediated healing of live autografts, we have developed a tissue-engineered periosteum (TEP) to improve allograft healing. The TEP comprises cell-degradable poly(ethylene glycol) hydrogels encapsulating mouse mesenchymal stem cells and osteoprogenitor cells to mimic the periosteal cell population. Despite improvements in allograft healing, several limitations were observed using the TEP, specifically the modulation of host tissue infiltration and remodeling to support graft-localized vascular volume and callus bridging. Therefore, hydrogel biochemical cues were incorporated into TEP to enable cell-matrix interactions and remodeling critical for tissue infiltration. Adhesive peptide functionalization (RGD, YIGSR, and GFOGER) and enzymatic degradation rate (GPQGIWGQ, IPESLRAG, and VPLSLYSG) were screened using an in vitro 3D cell spheroid assay and design of experiments (DOE) to identify hydrogels that best supported tissue infiltration and integration. DOE analysis of various adhesive peptide combinations was used to optimize functionalization, revealing that individual RGD-functionalization and GFOGER-functionalization maximized in vitro cell infiltration. RGD and GFOGER hydrogels were then investigated in vivo as TEP (RGD-TEP and GFOGER-TEP, respectively) to evaluate the effect of hydrogel functionalization on TEP-mediated allograft healing in a murine femur defect model. RGD- and GFOGER-TEP promoted bone graft healing, with both groups exhibiting a 1.9-fold increase in bone callus volume over unmodified allografts at 3 weeks post-implantation. RGD-TEP promoted more significant bone tissue development, but GFOGER-TEP promoted greater torsional biomechanics over time. The few differences observed between TEP groups suggest hydrogel functionalization has a limited effect on TEP-mediated healing, with cell delivery via the TEP enough to improve bone regeneration. Future studies aim to investigate additional adhesive peptides with diverse combinations to identify potential synergies between adhesive peptides to promote TEP-mediated bone allograft healing.

Disruption of photoreceptor-retinal pigment epithelium (RPE) interface with loss of photoreceptor outer segments (POSs) in the retina is a pathological hallmark of several neurodegenerative and retinal diseases including lysosomal storage disorder's like CLN3 disease. However, the retina is a functional composite in vivo; and in vitro stem cell models of retina that enable investigation of the photoreceptor-RPE interface in healthy and diseased retina are lacking. Here, we developed a 3D human pluripotent stem cell (hPSC)-derived retina model to investigate the photoreceptor-RPE interface in healthy and disease tissue. Using this 3D hPSC retina model, we demonstrated that the most common disease causing CLN3 mutation ( CLN3 Δ ex7-8 ) leads to reduced levels of acid ceramidase (AC) and consequently altered sphingolipid metabolism and signaling and POS loss in CLN3 disease. Consistent with the 3D hPSC retina model, altered sphingolipid metabolism and signaling coincided with POS loss in a large animal model of CLN3 disease, CLN3 miniswine. Therapeutically, recombinant human acid ceramidase (rhAC) targeted both altered sphingolipid metabolism and retina degeneration in the CLN3 hPSC retina model and the CLN3 miniswine eye. These findings demonstrate a proof-of-concept that rhAC can rescue disease phenotype in a large animal model of CLN3 disease and suggest that rhAC could be a therapeutic approach for CLN3 disease. One Sentence Summary:Acid ceramidase deficiency and consequently altered sphingolipid signaling promotes disease phenotype(s) in a lysosomal storage disorder, CLN3 disease.

Novel approaches are needed to study relationships between oral biofilm strains, enable three-dimensional oral biofilm deposition, and hasten the rigor and pace of basic and translational biofilm studies. Previously, 3D-bioprinters were leveraged to deposit spatially patterned biofilms onto sugar-rich agar surfaces to study how the underlying spatial organization of various microbes impacts biofilm persistence and virulence. Herein, we have developed a new method to adapt this process from limited, soft agar surfaces to biomimetic solid substrates submerged in aqueous solutions for studying oral biofilms in vitro. Streptococcus mutans UA159 was used to compare standard in vitro biofilm development with our new 3D-printed bio-ink hydrogels on hydroxyapatite disks, which mimic tooth surfaces. Biofilms formed using the bio-ink methodology showed minimal quantitative differences in virulence factors, including environmental pH, biomass, and cell density, compared to biofilms formed using the standard in vitro methodology. The bio-ink technique resulted in higher exopolysaccharide deposition, a key virulence factor for biofilm cohesion and protection, as well as more homogeneous spatial distribution of bacterial microcolonies. Our newly developed technique produces 3D-printable model biofilms that match the virulence benchmarks of the standard method, opening possibilities to print biofilms onto any substrate and a new way to study multidimensional biofilm dynamics.IMPORTANCEDental caries is the most common oral disease caused by biofilms in humans with cost limitations. Changes in the human diet have increased the exposure to sugar-rich processed food, increasing the incidence and severity of dental caries and creating greater rationale for understanding biofilm deposition, microbial interactions, and maintenance of quiescence of the oral microbiota. Recent 3D-printing techniques have been leveraged to develop the first model biofilms, providing spatial control over microbe deposition and enabling unprecedented investigation of the impact of cell-cell interactions and spatial organizationupon biofilm persistence, sensitivity to drugs, and virulence. Here, we have developed new methods to extend bioprinting to oral biofilms using cariogenic Streptococcus mutans. Our technique is an attempt to establish an alternative method for oral biofilm formation in vitro that uses 3D-printing tools, preserving the virulence of standard in vitro biofilms while amplifying the availability and versatility of methods for understanding the microbiome.

Most head and neck cancer patients treated with ionizing radiation loose salivary gland function. Patients with decreased saliva have trouble eating, speaking and are predisposed to oral infections and tooth decay. Amifostine is the only FDA approved drug to prevent radiation-induced hyposalivation. However, it has intolerable side-effects that limit its use, motivating the discovery of alternative therapeutics. We leveraged our salivary gland tissue chip platform for high-content drug discovery that we developed using submandibular gland tissue from female SKH1 hairless mice, backcrossed 6 generations with C57BL/6 J mice. We developed in-chip assays to quantify reduced glutathione and cellular senescence, which are accepted biomarkers of radiation damage. We validated radioprotection using WR-1065, the active form of Amifostine and tested other reported radioprotective drugs including Edaravone, Tempol, N-acetylcysteine, Rapamycin, Ex-Rad, and Palifermin. Next, a Selleck Chemicals library of FDA-approved drugs was screened for radioprotection. Lead hits were tested in mouse models. We identify 25 candidate compounds and down-select them using EC50 values and published pharmacologic data. This lead us to test Phenylbutazone (an anti-inflammatory), Enoxacin (a fluoroquinolone antibiotic), and Doripenem (a carbapenem antibiotic) for in vivo radioprotection in mice. Results confirm that these three drugs exhibit radioprotection equivalent to Amifostine but with superior EC50 values, ranging from 140 to 6900-fold lower values. This body of work demonstrates the development and validation of assays using a tissue chip platform for high-content drug screening and the successful discovery and in vivo validation of candidate radioprotective drugs with non-antioxidant primary modes of action. These results point to possible unknown mechanisms of radioprotection. These drugs can be developed to improve radioprotection efficacy and clinical administration without adverse side-effects. Salivary glands are often damaged in patients undergoing radiation therapy to treat head and neck cancers. Lack of saliva results in a poor quality of life for these patients. Currently there are no good drugs that patients can take to protect their salivary glands from radiation damage. We used a system that models the salivary gland called a tissue chip to test a large panel of drugs to see if they can prevent damage by radiation. We identified several drug candidates that could protect mouse salivary glands from radiation. These drugs should now be further investigated as they could potentially be used to protect the salivary glands of patients undergoing radiation therapy. Piraino, Chen, Mereness et al use a salivary gland tissue chip for high-content radioprotective drug discovery. Three drugs are identified that exhibit higher potency and equivalent radioprotection to Amifostine, the only FDA approved drug that is rarely used clinically due to severe side effects and lack of efficacy.

Solid phase peptide synthesis (SPPS) techniques are critical for developing and using peptides in various biomedical applications. However, typical synthesis routes used in SPPS are either resource-intensive (e.g., with automated synthesis or commercial services) or time-consuming (e.g., with manual benchtop synthesis). Here, a rapid manual synthesis method was developed to produce up to 8 peptides with fast cycle times simultaneously. Peptides synthesized manually were of equivalent or superior quality to those produced by in-house microwave-assisted automated peptide synthesis, with higher average crude purity of 70% compared to 50%. The method significantly reduced synthesis time, enabling the parallel coupling of up to 8 amino acids simultaneously in 15-20 min, as opposed to traditional benchtop peptide synthesis, which requires 80-150 min per amino acid. This approach offers an intermediate throughput between milligram-scale libraries and gram-scale single peptide synthesis, enabling rapid iteration for novel peptide designs without the need for expensive automated systems. As a result, peptide modifications, including incorporation of unnatural amino acids, can be explored, accelerating the development of peptides for a wide range of applications.

Satisfactory healing following acute tendon injury is marred by fibrosis. Despite the high frequency of tendon injuries and poor outcomes, there are no pharmacological therapies in use to enhance the healing process. Moreover, systemic treatments demonstrate poor tendon homing, limiting the beneficial effects of potential tendon therapeutics. To address this unmet need, we leveraged our existing tendon healing spatial transcriptomics dataset and identified an area enriched for expression of Acp5 (TRAP) and subsequently demonstrated robust TRAP activity in the healing tendon. This unexpected finding allowed us to refine and apply our existing TRAP binding peptide (TBP) functionalized nanoparticle (NP) drug delivery system (DDS) to facilitate improved delivery of systemic treatments to the healing tendon. To demonstrate the translational potential of this DDS, we delivered niclosamide (NEN), an S100a4 inhibitor. While systemic delivery of free NEN did not alter healing, TBP-NP NEN enhanced both functional and mechanical recovery, demonstrating the translational potential of this approach to enhance the tendon healing process.

Head and neck cancers (HNCs) rank as the sixth most common cancer globally and result in over 450 000 deaths annually. Despite considerable advancements in diagnostics and treatment, the 5-year survival rate for most types of HNCs remains below 50%. Poor prognoses are often attributed to tumor heterogeneity, drug resistance, and immunosuppression. These characteristics are difficult to replicate using in vitro or in vivo models, culminating in few effective approaches for early detection and therapeutic drug development. Organs-on-a-chip offer a promising avenue for studying HNCs, serving as microphysiological models that closely recapitulate the complexities of biological tissues within highly controllable microfluidic platforms. Such systems have gained interest as advanced experimental tools to investigate human pathophysiology and assess therapeutic efficacy, providing a deeper understanding of cancer pathophysiology. This review outlines current challenges and opportunities in replicating HNCs within microphysiological systems, focusing on mimicking the soft, glandular, and hard tissues of the head and neck. We further delve into the major applications of organ-on-a-chip models for HNCs, including fundamental research, drug discovery, translational approaches, and personalized medicine. This review emphasizes the integration of organs-on-a-chip into the repertoire of biological model systems available to researchers. This integration enables the exploration of unique aspects of HNCs, thereby accelerating discoveries with the potential to improve outcomes for HNC patients.

Nanoparticle drug delivery systems (NP DDS) have proven to be tremendously impactful for delivering therapeutic agents in cancer treatments, vaccinations, gene therapy, diagnostics, and enabled new agents such as RNA therapeutics. However, the exposure of NP DDS to biological milieus leads to the rapid adsorption of proteins and other molecules, forming a proteinaceous corona that obscures NP surface characteristics and controls the biological interactions of the NP DDS. This corona often potentiates phagocytic uptake, hindering the efficacy of targeting groups, and reducing NP drug delivery efficacy. Surface modifications to reduce protein adsorption can overcome these deleterious effects. Still, the repertoire of modifications is limited to poly(ethylene glycol) (PEG)and synthetic zwitterionic polymers. However, these anti-fouling strategies lack tunability, have off-target immunological effects, and suffer from targeting-limited steric hindrance, altogether motivating the development of alternative approaches. Peptides can uniquely form many zwitterions and have shown promise in reducing and controlling NP protein coronas as a function of peptide sequence. However, the impact of ZIPs on the drug delivery properties of polymeric NPs has not been explored. In this work, diverse zwitterionic peptides (ZIPs) predicted computationally to reduce protein adsorption by assessing peptide-peptide β-strand interaction energies were conjugated to pH-responsive cationic NPs. The resulting ZIP-NP conjugates exhibited up to 88% reduced protein adsorption versus unfunctionalized NP, resulting in a range of siRNA-mediated gene knockdown, outcomes that correlate with interaction energies. These data suggest that interaction energy is a promising design parameter for ZIPs. ZIP-NP also exhibited sequence-dependent variations in cellular uptake and circulation half-life, indicating ZIP-NPs are suitable for tuning and improving NP drug delivery characteristics.

Silver Diamine Fluoride (SDF) is effective for arresting dental caries, presenting a valuable non-invasive treatment option in dentistry. Despite its therapeutic advantages, a significant drawback is the tooth discoloration that follows its application, which can affect patient acceptance. Addressing this aesthetic concern without diminishing the treatment's efficacy remains challenging in dental practice. This study explores strategies to improve the aesthetic outcomes of SDF treatments. This in vitro study assessed the efficacy of Zinc in reducing SDF-induced discoloration on dentin blocks and examined its impact on the physical properties of dentin, including hardness and roughness. Dentin blocks were pre-treated with various concentrations of Zinc, followed by SDF application. Color changes were analyzed using Image J software, and cytotoxicity was evaluated using the CytoTox-ONE™ Homogeneous Membrane Integrity Assay. Dentin surface characteristics, including micro-hardness and roughness, were assessed using scanning electron microscopy. The study results revealed a dose-dependent efficacy of Zinc in reducing discoloration caused by SDF on dentin, with higher Zinc concentrations showing better improvement in color outcomes. The application of a 20 M Zinc solution prior to SDF treatment significantly reduced discoloration compared to SDF alone, measured on day 14. Additionally, no significant changes in the hardness or roughness of etched dentin were observed in Zinc + SDF group compared to the SDF alone group. Zinc treatments demonstrated a desirable outcome on mucosal cytotoxicity, comparable to that of the negative control. Zinc significantly reduced SDF-induced tooth discoloration in a dose-dependent manner without affecting the etched dentin's micro-hardness and roughness, potentially improving patient acceptance, especially in visible areas of the mouth. Further research is warranted to validate the effectiveness of this zinc-enhanced treatment protocol in vivo. Zinc-enhanced SDF treatments could enhance patient acceptance, especially in visible areas of the mouth, offering an improved option for caries management.

Designing targeted drug delivery systems to effectively treat bone diseases ranging from osteoporosis to nonunion bone defects remains a significant challenge. Previously, nanoparticles (NPs) self-assembled from diblock copolymers of poly(styrene-alt-maleic anhydride)-b-poly(styrene) (PSMA-b-PS) delivering a Wnt agonist were shown to effectively target bone and improve healing via the introduction of a peptide with high affinity to tartrate-resistant acid phosphatase (TRAP), an enzyme deposited by the osteoclasts during bone remodeling. Despite these promising results, the underlying biological factors governing targeting and subsequent drug delivery system (DDS) design parameters have not been examined to enable the rational design to improve bone selectivity. Therefore, this work investigated the effect of target ligand density, the treatment window after injury, specificity of TRAP binding peptide (TBP), the extent of TRAP deposition, and underlying genetic factors (e.g., mouse strain differences) on TBP-NP targeting. Data based on in vitro binding studies and in vivo biodistribution analyses using a murine femoral fracture model suggest that TBP-NP-TRAP interactions and TBP-NP bone accumulation were ligand-density-dependent; in vitro, TRAP affinity was correlated with ligand density up to the maximum of 200,000 TBP ligands/NP, while NPs with 80,000 TBP ligands showed 2-fold increase in fracture accumulation at day 21 post injury compared with that of untargeted or scrambled controls. While fracture accumulation exhibited similar trends when injected at day 3 compared to that at day 21 postfracture, there were no significant differences observed between TBP-functionalized and control NPs, possibly due to saturation of TRAP by NPs at day 3. Leveraging a calcium-depletion diet, TRAP deposition and TBP-NP bone accumulation were positively correlated, confirming that TRAP-TBP binding leads to TBP-NP bone accumulation in vivo. Furthermore, TBP-NP exhibited similar bone accumulation in both C57BL/6 and BALB/c mouse strains versus control NPs, suggesting the broad applicability of TBP-NP regardless of the underlying genetic differences. These studies provide insight into TBP-NP design, mechanism, and therapeutic windows, which inform NP design and treatment strategies for fractures and other bone-associated diseases that leverage TRAP, such as marrow-related hematologic diseases.

Michael addition between thiol- and maleimide-functionalized molecules is a long-standing approach used for bioconjugation, hydrogel crosslinking, and the functionalization of other advanced materials. While the simplicity of this chemistry enables facile synthesis of hydrogels, network degradation is also desirable in many instances. Here, the susceptibility of thiol-maleimide bonds to radical-mediated degradation is reported. Irreversible degradation in crosslinked materials is demonstrated using photoinitiated and chemically initiated radicals in hydrogels and linear polymers. The extent of degradation is shown to be dependent on initiator concentration. Using a model linear polymer system, the radical-mediated mechanism of degradation is elucidated, in which the thiosuccinimide crosslink is converted to a succinimide and a new thioether formed with an initiator fragment. Using laser stereolithography, high-fidelity spatiotemporal control over degradation in crosslinked gels is demonstrated. Ultimately, this work establishes a platform for controllable, radical-mediated degradation in thiol-maleimide hydrogels, further expanding their versatility as functional materials.

Fracture healing is a complex interplay of molecular and cellular mechanisms lasting from days to weeks. The inflammatory phase is the first stage of fracture healing and is critical in setting the stage for successful healing. There has been growing interest in exploring the role of the immune system and novel therapeutic strategies, such as nanoparticle drug delivery systems in enhancing fracture healing. Advancements in nanotechnology have revolutionized drug delivery systems to the extent that they can modulate immune response during fracture healing by leveraging unique physiochemical properties. Therefore, understanding the intricate interactions between nanoparticle-based drug delivery systems and the immune response, specifically macrophages, is essential for therapeutic efficacy. This review provides a comprehensive overview of the relationship between the immune system and nanoparticles during fracture healing. Specifically, we highlight the influence of nanoparticle characteristics, such as size, surface properties, and composition, on macrophage activation, polarization, and subsequent immune responses. Impact statement: This review provides valuable insights into the interplay between fracture healing, the immune system, and nanoparticle-based drug delivery systems. Understanding nanoparticle-macrophage interactions can advance the development of innovative therapeutic approaches to enhance fracture healing, improve patient outcomes, and pave the way for advancements in regenerative medicine.

In many tissues, including musculoskeletal tissues such as tendon, systemic delivery typically results in poor targeting of free drugs. Hence, we previously developed a targeted drug delivery nanoparticle (NP) system for tendon healing, leveraging a tartrate resistant acid phosphatase (TRAP) binding peptide (TBP) ligand. The greatest tendon targeting was observed with NPs functionalized with 30 000 TBP ligands per NP at day 7 during the proliferative healing phase, relative to the inflammatory (day 3) and early remodeling (day 14) phases of healing. Nevertheless, TRAP activity varies throughout healing and, therefore, may offer an opportunity for optimizing temporal therapeutic targeting through multivalent interactions. Hence, in this study, we hypothesized that the ligand density (9000-55,000 TBPs per NP) can optimize tendon accumulation on the basis of variable TRAP levels. The multivalent nanoparticles were loaded with three different fluorophores. In vitro, the ligand density and fluorophore had no effect on the physicochemical properties of the NPs, including size, charge, polydispersity index, or dye loading efficiency; however, the TRAP binding affinity correlated positively with the ligand density. In vivo, the ligand density correlated positively with NP homing and retention in the tendon, establishing opportunities to leverage ligand density for tendon targeting across the tendon healing cascade, during aging, and in other tendon pathologies, including tendinopathies.