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Following injury, the oral mucosa undergoes a complex sequences of biological healing processes to restore homeostasis. While general similarities exist, there are marked differences in the genomics and kinetics of wound healing between the oral cavity and cutaneous epithelium. The lack of successful therapy for oral mucosal wounds has influenced clinicians to explore alternative treatments and potential autotherapies to enhance intraoral healing. The present in-depth review discusses current gold standards for oral mucosal wound healing and compares endogenous factors that dictate the quality of tissue remodeling. We conducted a review of the literature on in vivo oral wound healing models and emerging regenerative therapies published during the past twenty years. Studies were evaluated by injury models, therapy interventions, and outcome measures. The success of therapeutic approaches was assessed, and research outcomes were compared based on current hallmarks of oral wound healing. By leveraging therapeutic advancements, particularly within in cell-based biomaterials and immunoregulation, there is great potential for translational therapy in oral tissue regeneration.

Dysfunction of the lymphatic system following injury, disease, or cancer treatment can lead to lymphedema, a debilitating condition with no cure. Despite the various physical therapy and surgical options available, most treatments are palliative and fail to address the underlying lymphatic vascular insufficiency driving lymphedema progression. Stem cell therapy provides a promising alternative in the treatment of various chronic diseases with a wide range of therapeutic effects that reduce inflammation, fibrosis, and oxidative stress, while promoting lymphatic vessel (LV) regeneration. Specifically, stem cell transplantation is suggested to promote LV restoration, rebuild lymphatic circulation, and thus potentially be utilized towards an effective lymphedema treatment. In addition to stem cells, studies have proposed the administration of vascular endothelial growth factor C (VEGFC) to promote lymphangiogenesis and decrease swelling in lymphedema.Here, we seek to combine the benefits of stem cell therapy, which provides a cellular therapeutic approach that can respond to the tissue environment, and VEGFC administration to restore lymphatic drainage.Specifically, we engineered mesenchymal stem cells (MSCs) to overexpress VEGFC using a lentiviral vector (hVEGFC MSC) and investigated their therapeutic efficacy in improving LV function and tissue swelling using near infrared (NIR) imaging, and lymphatic regeneration in a single LV ligation mouse tail lymphedema model.First, we showed that overexpression of VEGFC using lentiviral transduction led to an increase in VEGFC protein synthesis in vitro. Then, we demonstrated hVEGFC MSC administration post-injury significantly increased the lymphatic contraction frequency 14-, 21-, and 28-days post-surgery compared to the control animals (MSC administration) in vivo, while also reducing tail swelling 28-days post-surgery compared to controls.Our results suggest a therapeutic potential of hVEGFC MSC in alleviating the lymphatic dysfunction observed during lymphedema progression after secondary injury and could provide a promising approach to enhancing autologous cell therapy for treating lymphedema.

Successful bone healing in severe trauma depends on early revascularization to restore oxygen, nutrient, growth factor, and progenitor cell supply to the injury. Therapeutic angiogenesis strategies have therefore been investigated to promote revascularization following severe bone injuries; however, results have been inconsistent. This is the first study investigating the effects of dual angiogenic growth factors (VEGF and PDGF) with low-dose bone morphogenetic protein-2 (BMP-2; 2.5 µg) on bone healing in a clinically challenging composite bone-muscle injury model. Our hydrogel-based delivery systems demonstrated a more than 90% protein entrapment efficiency and a controlled simultaneous release of three growth factors over 28 days. Co-stimulation of microvascular fragment constructs with VEGF and PDGF promoted vascular network formation in vitro compared to VEGF or PDGF alone. In an in vivo model of segmental bone and volumetric muscle loss injury, combined VEGF (5 µg) and PDGF (7.5 µg or 15 µg) delivery with a low dose of BMP-2 significantly enhanced regeneration of vascularized bone compared to BMP-2 treatment alone. Notably, the regenerated bone mechanics reached ~60% of intact bone, a value that was previously only achieved by delivery of high-dose BMP-2 (10 µg) in this injury model. Overall, sustained delivery of VEGF, PDFG, and BMP-2 is a promising strategy to promote functional vascularized bone tissue regeneration following severe composite musculoskeletal injury. Although this study is conducted in a clinically relevant composite injury model in rats using a simultaneous release strategy, future studies are necessary to test the regenerative potential of spatiotemporally controlled delivery of triple growth factors on bone healing using large animal models. Statement of significance Volumetric muscle loss combined with delayed union or non-union bone defect causes deleterious effects on bone regeneration even with the supplementation of bone morphogenetic protein-2 (BMP-2). In this study, the controlled delivery of dual angiogenic growth factors (vascular endothelial growth factor [VEGF] + Platelet-derived growth factor [PDGF]) increases vascular growth in vitro. Co-delivering VEGF+PDGF significantly increase the bone formation efficacy of low-dose BMP-2 and improves the mechanics of regenerated bone in a challenging composite bone-muscle injury model.

Background: Intra-articular injections of human mesenchymal stromal cells (hMSCs) have shown promise in slowing cartilage degradation in posttraumatic osteoarthritis (PTOA). Clinical use of cell therapies for osteoarthritis has accelerated in recent years without sufficient scientific evidence defining best-use practices. Common recommendations advise patients to avoid nonsteroidal anti-inflammatory drug (NSAID) use before and after cell injection over concerns that NSAIDs may affect therapeutic efficacy. Recommendations to restrict NSAID use are challenging for patients, and it is unclear if patients are compliant. Hypothesis: NSAIDs will reduce the efficacy of hMSC therapy in treating a preclinical model of PTOA. Study Design: Controlled laboratory study. Methods: Lewis rats underwent medial meniscal transection (MMT) surgery to induce PTOA or a sham (sham group) surgery that did not progress to PTOA. Rats received naproxen solution orally daily before (Pre-NSAID group) or after (Post-NSAID group) hMSC treatment, throughout the course of the experiment (Full-NSAID group), or received hMSCs without NSAIDs (No NSAID). Cartilage morphology and composition were quantified using contrast-enhanced micro–computed tomography and histology. Pain (secondary allodynia) was measured using a von Frey filament. Results: Injection of hMSCs attenuated cartilage degeneration associated with MMT. hMSCs prevented proteoglycan loss, maintained smooth cartilage surfaces, reduced cartilage lesions, reduced mineralized osteophyte formation, and reduced pain by week 7. The Pre-NSAID group had decreased proteoglycan levels compared with the hMSC group, although there were no other significant differences. Thus, pretreatment with NSAIDs had minimal effects on the therapeutic benefits of hMSC injections. The Post-NSAID and Full-NSAID groups, however, exhibited significantly worse osteoarthritis than the hMSC-only group, with greater proteoglycan loss, surface roughness, osteophyte volume, and pain. Conclusion: Use of NSAIDs before hMSC injection minimally reduced the therapeutic benefits for PTOA, which included preservation of cartilage surface integrity as well as a reduction in osteophytes. Use of NSAIDs after injections, however, substantially reduced the therapeutic efficacy of cellular treatment. Clinical Relevance: Our data support the clinical recommendation of avoiding NSAID use after hMSC injection but suggest that using NSAIDs before treatment may not substantially diminish the therapeutic efficacy of cell treatment.

IntroductionOrthopaedic trauma and fracture care commonly cause perioperative anaemia and associated functional iron deficiency due to a systemic inflammatory state. Modern, strict transfusion thresholds leave many patients anaemic; managing this perioperative anaemia is an opportunity to impact outcomes in orthopaedic trauma surgery. The primary outcome of this pilot study is feasibility for a large randomised controlled trial (RCT) to evaluate intravenous iron therapy (IVIT) to improve patient well-being following orthopaedic injury. Measurements will include rate of participant enrolment, screening failure, follow-up, missing data, adverse events and protocol deviation.Methods and analysisThis single-centre, pilot, double-blind RCT investigates the use of IVIT for acute blood loss anaemia in traumatically injured orthopaedic patients. Patients are randomised to receive either a single dose infusion of low-molecular weight iron dextran (1000 mg) or placebo (normal saline) postoperatively during their hospital stay for trauma management. Eligible subjects include adult patients admitted for lower extremity or pelvis operative fracture care with a haemoglobin of 7–11 g/dL within 7 days postoperatively during inpatient care. Exclusion criteria include history of intolerance to intravenous iron supplementation, active haemorrhage requiring ongoing blood product resuscitation, multiple planned procedures, pre-existing haematologic disorders or chronic inflammatory states, iron overload on screening or vulnerable populations. We follow patients for 3 months to measure the effect of iron supplementation on clinical outcomes (resolution of anaemia and functional iron deficiency), patient-reported outcomes (fatigue, physical function, depression and quality of life) and translational measures of immune cell function.Ethics and disseminationThis study has ethics approval (Oregon Health & Science University Institutional Review Board, STUDY00022441). We will disseminate the findings through peer-reviewed publications and conference presentations.Trial registration numberNCT05292001; ClinicalTrials.gov.

The lymphatic system has been proposed to play a crucial role in preventing the development and progression of osteoarthritis (OA). As OA develops and progresses, inflammatory cytokines and degradation by-products of joint tissues build up in the synovial fluid (SF) providing a feedback system to exacerbate disease. The lymphatic system plays a critical role in resolving inflammation and maintaining overall joint homeostasis; however, there is some evidence that the lymphatics can become dysfunctional during OA. We hypothesized that the functional mechanics of lymphatic vessels (LVs) draining the joint could be directly compromised due to factors within SF derived from osteoarthritis patients (OASF). Here, we utilized OASF and SF derived from healthy (non-OA) individuals (healthy SF (HSF)) to investigate potential effects of SF entering the draining lymph on migration of lymphatic endothelial cells (LECs) in vitro, and lymphatic contractile activity of rat femoral LVs (RFLVs) ex vivo. Dilutions of both OASF and HSF containing serum resulted in a similar LEC migratory response to the physiologically endothelial basal medium-treated LECs (endothelial basal medium containing serum) in vitro. Ex vivo, OASF and HSF treatments were administered within the lumen of isolated LVs under controlled pressures. OASF treatment transiently enhanced the RFLVs tonic contractions while phasic contractions were significantly reduced after 1 h of treatment and complete ceased after overnight treatment. HSF treatment on the other hand displayed a gradual decrease in lymphatic contractile activity (both tonic and phasic contractions). The observed variations after SF treatments suggest that the pump function of lymphatic vessel draining the joint could be directly compromised in OA and thus might present a new therapeutic target.

Significance: Changes in interstitial fluid clearance are implicated in many diseases. Using near-infrared (NIR) imaging with properly sized tracers could enhance our understanding of how venous and lymphatic drainage are involved in disease progression or enhance drug delivery strategies. Aim: We investigated multichromatic NIR imaging with multiple tracers to assess in vivo microvascular clearance kinetics and pathways in different tissue spaces. Approach: We used a chemically inert IR Dye 800CW (D800) to target venous capillaries and a purified conjugate of IR dye 680RD with 40 kDa PEG (P40D680) to target lymphatic capillaries in vivo. Optical imaging settings were validated and tuned in vitro using tissue phantoms. We investigated multichromatic NIR imaging’s utility in two in vivo tissue beds: the mouse tail and rat knee joint. We then tested the ability of the approach to detect interstitial fluid perturbations due to exercise. Results: In an in vitro simulated tissue environment, free dye and PEG mixture allowed for simultaneous detection without interference. In the mouse tail, co-injected NIR tracers cleared from the interstitial space via distinct routes, suggestive of lymphatic and venous uptake mechanisms. In the rat knee, we determined that exercise after injection transiently increased lymphatic drainage as measured by lower normalized intensity immediately after exercise, whereas exercise pre-injection exhibited a transient delay in clearance from the joint. Conclusions: NIR imaging enables simultaneous imaging of lymphatic and venous-mediated fluid clearance with great sensitivity and can be used to measure temporal changes in clearance rates and pathways.

Osteoarthritis (OA) is a widespread disease that continues to lack approved and efficacious treatments that modify disease progression. Micronized dehydrated human amnion/chorion membrane (μ-dHACM) has been shown to be effective in reducing OA progression, but many of the engineering design parameters have not been explored. The objectives of this study were to characterize the particle size distributions of two μ-dHACM formulations and to investigate the influence of these distributions on the

Background: Our understanding of the biology of ankle arthrodesis is based largely on work in spine and long bone animal models. However, the local soft tissue and vascular anatomy of the foot and ankle is different from that of the spine. Accordingly, the objective of this study was to develop a small animal ankle arthrodesis model. Methods: A total of 12 Lewis rats successfully underwent ankle arthrodesis with stabilization consisting of a single Kirschner wire across the prepared tibiotalar joint. Based on high nonunion rates with this initial procedure, a modification was made consisting of a second pin crossing the joint. A total of 6 rats underwent the second procedure. Radiographs were taken postoperatively and in 2-week intervals up to 10 weeks. Micro computed tomography (µCT) and histological analysis was conducted at 10 weeks to assess the fusion mass. Osseous bridging of greater than 50% across the tibiotalar joint was deemed a successful fusion. Results: µCT analysis determined that 11 of the 12 rats in the single-pin cohort developed nonunions (8.3% fusion rate). In the dual-pin cohort, all 6 animals successfully fused (100% fusion rate). Histological analysis supported the radiographic imaging conclusions. Conclusion: While the initial procedure had a high nonunion rate, enhancing the stability of the fixation greatly increased the union rate. Clinical Relevance: The present work demonstrates the first reliable small animal ankle arthrodesis model. We believe that this model can be used in the development of novel therapies aimed at decreasing complications and increasing fusion rates.

Clearance of particles from the knee is an essential mechanism to maintain healthy joint homeostasis and critical to the delivery of drugs and therapeutics. One of the limitations in developing disease modifying drugs for joint diseases, such as osteoarthritis (OA), has been poor local retention of the drugs. Enhancing drug retention within the joint has been a target of biomaterial development, however, a fundamental understanding of joint clearance pathways has not been characterized. We applied near-infrared (NIR) imaging techniques to assess size-dependent in vivo clearance mechanisms of intra-articular injected, fluorescently-labelled polyethylene glycol (PEG-NIR) conjugates. The clearance of 2 kDa PEG-NIR (τ = 171 ± 11 min) was faster than 40 kDa PEG-NIR (τ = 243 ± 16 min). 40 kDa PEG-NIR signal was found in lumbar lymph node while 2 kDa PEG-NIR signal was not. Thus, these two conjugates may be cleared through different pathways, i.e. lymphatics for 40 kDa PEG-NIR and venous for 2 kDa PEG-NIR. Endothelin-1 (ET-1), a potent vasoconstrictor of vessels, is elevated in synovial fluid of OA patients but, its effects on joint clearance are unknown. Intra-articular injection of ET-1 dose-dependently inhibited the clearance of both 2 kDa and 40 kDa PEG-NIR. ET-1 caused a 1.63 ± 0.17-fold increase in peak fluorescence for 2 kDa PEG-NIR and a 1.85 ± 0.15-fold increase for 40 kDa PEG-NIR; and ET-1 doubled their clearance time constants. The effects of ET-1 were blocked by co-injection of ET receptor antagonists, bosentan or BQ-123. These findings provide fundamental insight into retention and clearance mechanisms that should be considered in the development and delivery of drugs and biomaterial carriers for joint diseases. Statement of Significance This study demonstrates that in vivo knee clearance can be measured using NIR technology and that key factors, such as size of materials and biologics, can be investigated to define joint clearance mechanisms. Therapies targeting regulation of joint clearance may be an approach to treat joint diseases like osteoarthritis. Additionally, in vivo functional assessment of clearance may be used as diagnostics to monitor progression of joint diseases.

Oral cavity wound healing occurs in an environment that sustains ongoing physical trauma and is rich in bacteria. Despite this, injuries to the mucosal surface often heal faster than cutaneous wounds and leave less noticeable scars. Patients undergoing cleft palate repair have a high degree of wound healing complications with up to 60% experiencing oronasal fistula (ONF) formation. In this study, we developed a mouse model of hard palate mucosal injury, to study the endogenous injury response during oral cavity wound healing and ONF formation. Immunophenotyping of the inflammatory infiltrate following hard palate injury showed delayed recruitment of non-classical LY6Clo monocytes and failure to resolve inflammation. To induce a pro-regenerative inflammatory response, delivery of FTY720 nanofiber scaffolds following hard palate mucosal injury promoted complete ONF healing and was associated with increased LY6Clo monocytes and pro-regenerative M2 macrophages. Alteration in gene expression with FTY720 delivery included increased Sox2 expression, reduction in pro-inflammatory IL-1, IL-4 and IL-6 and increased pro-regenerative IL-10 expression. Increased keratinocyte proliferation during ONF healing was observed at day 5 following FTY720 delivery. Our results show that local delivery of FTY720 from nanofiber scaffolds in the oral cavity enhances healing of ONF, occurring through multiple immunomodulatory mechanisms. STATEMENT OF SIGNIFICANCE: Wound healing complications occur in up to 60% of patients undergoing cleft palate repair where an oronasal fistula (ONF) develops, allowing food and air to escape from the nose. Using a mouse model of palate mucosal injury, we explored the role of immune cell infiltration during ONF formation. Delivery of FTY720, an immunomodulatory drug, using a nanofiber scaffold into the ONF was able to attract anti-inflammatory immune cells following injury that enhanced the reepithelization process. ONF healing at day 5 following FTY720 delivery was associated with altered inflammatory and epithelial transcriptional gene expression, increased anti-inflammatory immune cell infiltration, and increased proliferation. These findings demonstrate the potential efficacy of immunoregenerative therapies to improve oral cavity wound healing.

Mesenchymal stromal cells (MSCs) have shown promise as osteoarthritis (OA) treatments; however, effective translation has been limited by high variability and heterogeneity of MSCs, suboptimal delivery strategies, and poor understanding of critical quality and potency attributes. Furthermore, most pre-clinical studies of MSC therapeutics for OA have focused on delaying OA development and not on treating established OA, which brings added clinical relevance. Thus, the objective of the current study was to assess the effects of sodium alginate microencapsulation on human MSC (hMSC) secretion of immunomodulatory cytokines in an OA microenvironment and therapeutic efficacy in treating established OA. A Medial Meniscal Transection (MMT) pre-clinical model of OA was implemented. Three weeks post-surgery, after OA was established, intra-articular injections of encapsulated hMSCs or nonencapsulated hMSCs were administered. Six weeks post-surgery, microstructural changes in the knee joint were quantified using microCT. Encapsulated hMSCs reduced articular cartilage degeneration and subchondral bone remodeling. A multiplexed immunoassay panel was used to profile the in vitro secretome of hMSCs in response to IL-1β. Nonencapsulated hMSCs showed an indiscriminate increase in all cytokines in response to IL-1β while encapsulated hMSCs showed a targeted secretory response with increased expression of pro-inflammatory (IL-1β, IL-6, IL-7, IL-8), anti-inflammatory (IL-1RA), and chemotactic (G-CSF, MDC, IP10) cytokines. These data show that sodium alginate microencapsulation can modulate hMSC paracrine signaling and enhance the therapeutic efficacy of the hMSCs in treating established OA. This cytokine profile provides a foundation for the identification of key factors affecting the overall potency of hMSC therapeutics for OA. STATEMENT OF SIGNIFICANCE: While there has been considerable interest in material based MSC encapsulation for treatment of OA, there are critical gaps in our translational understanding of these biomaterial-based technologies for OA. More specifically, previous studies have several important limitations: (1) they have been largely focused on preventing OA development, which limits their translational utility and (2) little prior work has been done to delineate potential routes/mechanisms by which material encapsulation alters MSC therapeutic action. In our manuscript, we aimed to fill these gaps in knowledge by testing the hypotheses that: (1) hMSC encapsulation can attenuate established disease progression, which is a more clinically relevant scenario and (2) hMSC encapsulation significantly changes the secreted paracrine factors from hMSCs.

Volumetric muscle loss (VML) results in permanent functional deficits and remains a substantial regenerative medicine challenge. A coordinated immune response is crucial for timely myofiber regeneration, however the immune response following VML has yet to be fully characterized. Here, we leveraged dimensionality reduction and pseudo-time analysis techniques to elucidate the cellular players underlying a functional or pathological outcome as a result of subcritical or critical VML in the murine quadriceps, respectively. We found that critical VML presented with a sustained presence of M2-like and CD206hiLy6Chi ‘hybrid’ macrophages whereas subcritical defects resolved these populations. These macrophage subsets may contribute to fibrogenesis in critical VML, especially in the presence of TGF-β. Furthermore, several T cell populations were significantly elevated in critical VML compared to subcritical injuries. Specifically, there was a significant increase of CD127+ T cells at days 3 and 7, and upregulated CD127 expression may indicate aberrant IL-7 signaling in critical VML. These results demonstrate a dysregulated immune response in critical VML that is unable to resolve the chronic inflammatory state and transition to a pro-regenerative microenvironment. These data provide important insights into potential therapeutic strategies which could reduce the immune cell burden and pro-fibrotic signaling characteristic of VML.

Osteoarthritis is a degenerative disease of synovial joints affecting all tissues, including the articular cartilage and underlying subchondral bone. Osteoarthritis animal models can recapitulate aspects of human disease progression and are commonly used to test the development of drugs, biomaterials, and cell therapies for treatment. The rat medial meniscus transection (MMT) model is a surgically induced post-traumatic osteoarthritis model and is one of the most commonly used models for therapeutic development; however, it is typically used to evaluate the efficacy of therapies to prevent disease development rather than testing the treatment of disease progression in already established disease. We describe herein, the qualitative and quantitative changes to articular cartilage, subchondral bone, and formation of osteophytes in rats at early-(3-weeks post-surgery), mid-(6-weeks post-surgery) and late-(12-weeks post-surgery) stages of osteoarthritis progression. Tibiae of MMT-operated animals showed loss of proteoglycan and fibrillation formation on articular cartilage surfaces as early as 3-weeks post-surgery. Using a contrast-enhanced μCT technique, quantitative, 3-dimensional analysis of the tibiae showed that the articular cartilage initially thickened at 3- and 6-weeks post-surgery and then decreased at 12-weeks post-surgery. This decrease in cartilage thickness corresponded with increased lesions in the articular cartilage, including fully degraded surfaces down to the subchondral bone layer. In this rat MMT model, subchondral bone thickening was significant at 6-weeks post-surgery and seem to follow cartilage damage. Osteophytes were found at 3-weeks post-surgery, which coincided with articular cartilage degradation. Cartilaginous osteophytes preceded mineralization suggesting that these marginal tissue growths most likely occurred through endochondral ossification. The use of the rat MMT model has predominantly been used out to 3-weeks, and most studies determine the effect of therapies to delay or prevent the onset of osteoarthritis. We provide evidence that an extension of the rat MMT model out to 6 and 12 weeks resembled more severe phenotypes of human osteoarthritis. The mid- to late-stages of rat MMT model can be used to evaluate the therapeutic efficacy of novel treatments to treat the progression of established disease — since patients typically present in the clinic when the disease is established and becomes symptomatic, thus evaluating the efficacy of new treatments at the late stage will be important for eventual clinical translation.

SignificanceChanges in interstitial fluid clearance are implicated in many diseases. Using NIR imaging with properly sized tracers could enhance our understanding of how venous and lymphatic drainage are involved in disease progression or enhance drug delivery strategies.AimWe investigated multichromatic NIR imaging with multiple tracers to assess in vivo microvascular clearance kinetics and pathways in different tissue spaces.ApproachWe used a chemically inert IR Dye 800CW (free dye) to target venous capillaries and a purified conjugate of IR Dye 680RD with a 40 kDa PEG (PEG) to target lymphatic capillaries in vivo. Optical imaging settings were validated and tuned in vitro using tissue phantoms. We investigated multichromatic NIR imaging’s utility in two in vivo tissue beds – the mouse tail and rat knee joint. We then tested the ability of the approach to detect interstitial fluid perturbations due to exercise.ResultsIn an in vitro simulated tissue environment, free dye and PEG mixture allowed for simultaneous detection without interference. Co-injected NIR tracers cleared from the interstitial space via distinct routes allowed assessment lymphatic and venous uptake in the mouse tail. We determined that exercise after injection transiently increased lymphatic drainage as measured by lower normalized intensity immediately after exercise, while exercise pre-injection exhibited a transient delay in clearance from the jointConclusionsNIR imaging enables of simultaneous imaging of lymphatic and venous-mediated fluid clearance with great sensitivity and can be used to measure transient changes in clearance rates and pathways.

Current strategies for regeneration of large bone fractures yield limited clinical success mainly due to poor integration and healing. Multidisciplinary approaches in design and development of functional tissue engineered scaffolds are required to overcome these translational challenges. Here, a new generation of hyperelastic bone (HB) implants, loaded with superparamagnetic iron oxide nanoparticles (SPIONs), are 3D bioprinted and their regenerative effect on large non-healing bone fractures is studied. Scaffolds are bioprinted with the geometry that closely correspond to that of the bone defect, using an osteoconductive, highly elastic, surgically friendly bioink mainly composed of hydroxyapatite. Incorporation of SPIONs into HB bioink results in enhanced bacteriostatic properties of bone grafts while exhibiting no cytotoxicity. In vitro culture of mouse embryonic cells and human osteoblast-like cells remain viable and functional up to 14 days on printed HB scaffolds. Implantation of damage-specific bioprinted constructs into a rat model of femoral bone defect demonstrates significant regenerative effect over the 2-week time course. While no infection, immune rejection, or fibrotic encapsulation is observed, HB grafts show rapid integration with host tissue, ossification, and growth of new bone. These results suggest a great translational potential for 3D bioprinted HB scaffolds, laden with functional nanoparticles, for hard tissue engineering applications.

Monoclonal antibodies (mAb) have had a transformative impact on treating cancers and immune-disorders. However, their use is limited by high development time and monetary cost, manufacturing complexities, suboptimal pharmacokinetics, and availability of disease-specific targets. To address some of these challenges, we developed an entirely synthetic, multivalent, Janus nanotherapeutic platform, called Synthetic Nanoparticle-Antibodies (SNAbs). SNAbs, with phage-display identified cell-targeting ligands on one “face”, and Fc-mimicking ligands on the opposite “face”, were synthesized using a custom, multi-step, solid-phase chemistry method. SNAbs efficiently targeted and depleted myeloid-derived immune-suppressor cells (MDSCs) from mouse-tumor and rat-trauma models, ex-vivo. Systemic injection of MDSC-targeting SNAbs efficiently depleted circulating MDSCs in a mouse triple-negative breast cancer model, enabling enhanced T cell and Natural Killer cell infiltration into tumors. Our results demonstrate that SNAbs are a versatile and effective functional alternative to mAbs, with advantages of a plug-and-play, cell-free manufacturing process, and high-throughput screening (HTS)-enabled library of potential targeting ligands.

Purpose: Mechanical loading of bone defects through rehabilitation is a promising approach to stimulate repair and reduce nonunion risk; however, little is known about how therapeutic mechanical stimuli modulate early-stage repair before mineralized bone formation. The objective of this study was to investigate the early effects of osteogenic loading on cytokine expression and angiogenesis during the first 3 weeks of BMP-2 mediated segmental bone defect repair. Materials and Methods: A rat model of BMP-2 mediated bone defect repair was subjected to an osteogenic mechanical loading protocol using ambulatory rehabilitation and a compliant, load-sharing fixator with an integrated implantable strain sensor. The effect of fixator load-sharing on local tissue strain, angiogenesis, and cytokine expression was evaluated. Results: Using sensor readings for local measurements of boundary conditions, finite element simulations showed strain became amplified in remaining soft tissue regions between 1 and 3 weeks (Week 3: load-sharing: ���1.89 �� 0.35% and load-shielded: ���1.38 �� 0.35% vs. Week 1: load-sharing: ���1.54 �� 0.17%; load-shielded: ���0.76 �� 0.06%). Multivariate analysis of cytokine arrays revealed that load-sharing significantly altered expression profiles in the defect tissue at 2 weeks compared to load-shielded defects. Specifically, loading reduced VEGF (p = 0.052) 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 at 3 weeks. Conclusions: Together, these results demonstrate that moderate ambulatory strains previously shown to stimulate bone regeneration significantly alter early angiogenic and cytokine signaling and may promote endochondral ossification.

Mesenchymal stromal cells (MSCs) have shown promise as osteoarthritis (OA) treatments; however, effective translation has been limited by high variability and heterogeneity of MSCs, suboptimal delivery strategies, and poor understanding of critical quality and potency attributes. Furthermore, most pre-clinical studies of MSC therapeutics for OA have focused on delaying OA development and not on treating established OA, which brings added clinical relevance. Thus, the objective of the current study was to assess the effects of biomaterial encapsulation on human MSC (hMSC) secretion of immunomodulatory cytokines in an OA microenvironment and therapeutic efficacy in treating established OA. A Medial Meniscal Transection (MMT) pre-clinical model of OA was implemented. Three weeks post-surgery, after OA was established, intra-articular injections of encapsulated hMSCs or nonencapsulated hMSCs were administered. Six weeks post-surgery, microstructural changes in the knee joint were quantified using microCT. Encapsulated hMSCs reduced articular cartilage degeneration and subchondral bone remodeling. A multiplexed immunoassay panel was used to profile the in vitro secretome of hMSCs in response to IL-1β. Nonencapsulated hMSCs showed an indiscriminate increase in all cytokines in response to IL-1β while encapsulated hMSCs showed a targeted secretory response with increased expression of pro-inflammatory (IL-1β, IL-6, IL-7, IL-8), anti-inflammatory (IL-1RA), and chemotactic (G-CSF, MDC, IP10) cytokines. These data show that biomaterial encapsulation can modulate hMSC paracrine signaling and enhance the therapeutic efficacy of the hMSCs in treating established OA. This cytokine profile provides a foundation for the identification of key factors affecting the overall potency of hMSC therapeutics for OA. Funding Information: This work was supported in part by VA (SPiRE) Grant [I21RX002372-01A1] from the United States (U.S.) Department of Veterans Affairs Rehabilitation Research and Development Service. The research was also supported in part by the DOD PRMRP Grant [PR171379] and PHS Grant [UL1TR000454] from the Clinical and Translational Science Award Program, National Institutes of Health, National Center for Advancing Translational Sciences. Declaration of Interests: There are no conflicts to declare. Ethics Approval Statement: All animal care and experiments were conducted in accordance with the institutional guidelines and approved by the Atlanta Veteran Affairs Medical Center (VAMC) with experimental procedures approved by the Atlanta VAMC Institutional Animal Care and Use Committee (IACUC).

The lymphatic system has been proposed to play a crucial role in the development and progression of osteoarthritis (OA). The synovial fluid (SF) of arthritic joints contains mediators of the inflammatory response and products of the injury to articular tissues, while lymphatic system plays a critical role in resolving inflammation and overall joint homeostasis. Despite the importance of both the lymphatic system and SF in OA disease, their relationship is still poorly understood. Here, we utilized SF derived from osteoarthritis patients (OASF) and healthy individuals (HSF) to investigate potential effects of SF on migration of lymphatic endothelial cells (LECs) in vitro, and lymphatic contractility of femoral lymphatic vessels (LVs) ex vivo. Both OASF and HSF treatments led to an increased migratory response in vitro compared to LECs treatment with media without serum. Ex vivo, both OASF and HSF treatments to the lumen of isolated LVs led to significant differences in the tonic and phasic contractions and these observations were dependent on the SF treatment time. Specifically, OASF treatment transiently enhanced the RFLVs tonic contractions. Regarding the phasic contractions, OASF generated either an abrupt reduction after 1 hr of treatment or a complete cease of contractions after an overnight treatment, while HSF treatment displayed a gradual decrease in lymphatic contractility. The observed variations after SF treatments suggest that the pump function of lymphatic vessel draining the joint could be directly compromised in OA and thus might present a new therapeutic target.

Purpose Cleft palate repair surgeries lack a regenerative reconstructive option and, in many cases, develop complications including oronasal fistula (ONF). Our group has developed a novel murine phenocopy of ONF to study the oral cavity wound healing program. Using this model, our team previously identified that delivery of FTY720 on a nanofiber scaffold had a unique immunomodulatory effect directing macrophages and monocytes into a pro-regenerative state during ONF healing. Here, the objective of this study was to determine the effects of local biomaterial-based FTY720 delivery in the ONF model on the early bulk gene expression and neutrophil phenotypic response within the regenerating tissue. Methods Using a mouse model of ONF formation, a palate defect was created and was treated with FTY720 nanofiber scaffolds or (blank) vehicle control nanofibers. At 1 and 3 days post-implantation, ONF oral mucosal tissue from the defect region was collected for RNA sequencing analysis or flow cytometry. For the RNA-seq expression profiling, intracellular pathways were assessed using the KEGG Pathway database and Gene Ontology (GO) Terms enrichment interactive graph. To assess the effects of FTY720 on different neutrophil subpopulations, flow cytometry data was analyzed using pseudotime analysis based on Spanning-tree Progression Analysis of Density-normalized Events (SPADE). Results RNA sequencing analysis of palate mucosa injured tissue identified 669 genes that were differentially expressed (DE) during the first 3 days of ONF wound healing after local delivery of FTY720, including multiple genes in the sphingolipid signaling pathway. Evaluation of the DE genes at the KEGG Pathway database also identified the inflammatory immune response pathways (chemokine signaling, cytokine-cytokine receptor interaction, and leukocyte transendothelial migration), and the Gene Ontology enrichment analysis identified neutrophil chemotaxis and migration terms. SPADE dendrograms of CD11b+Ly6G+ neutrophils at both day 1 and day 3 post-injury showed significantly distinct subpopulations of neutrophils in oral mucosal defect tissue from the FTY720 scaffold treatment group compared to the vehicle control group (blank). Increased expression of CD88 and Vav1, among other genes, were found and staining of the ONF area demonstrated increased VAV1 staining in FTY720‐treated healing oral mucosa. Conclusion Treatment of oral mucosal defects using FTY720 scaffolds is a promising new immunotherapy to improve healing outcomes and reducing ONF formation during cleft palate surgical repair. Local delivery of FTY720 nanofiber scaffolds during ONF healing significantly shifted early gene transcription associated with immune cell recruitment and modulation of the immune microenvironment results in distinct neutrophil subpopulations in the oral mucosal defect tissue that provides a critical shift toward pro-regenerative immune signaling.

ObjectiveOsteoarthritis (OA) is a chronic degenerative disease of the joints characterized by articular cartilage degradation. While there are clear sex differences in OA development in humans, most pre-clinical research has been conducted solely in male animals thus limiting the ability of these findings to be generalized to both sexes in the context of this disease. The objective of this study was to determine if sex impacts the progression and severity of OA in the rat medial meniscal tear (MMT) preclinical animal model used to surgically induce OA. It was hypothesized that differences would be observed between males and females following MMT surgery.DesignA MMT model was employed in male and female Lewis rats to induce OA. Animals were euthanized 3 weeks post-surgery and EPIC-μCT was used to quantitatively evaluate articular cartilage structure and composition, osteophyte volumes and subchondral bone structure.ResultsQuantitative analysis of the medial 1/3 articular cartilage via EPIC-μCT showed increased cartilage thickness and proteoglycan loss in the MMT of both sexes, when compared to sham. Additionally, both male and female animals in the MMT group had increased subchondral bone mineral density and larger total osteophyte volumes due to MMT.ConclusionThese data demonstrate that OA can be induced in both sexes using the rat MMT model. Moving forward, adding sex as a factor in preclinical OA studies should be standard practice in pre-clinical studies in order to elucidate more inclusive and translatable results into the clinic.

Extracellular matrix deformation temporally regulates angiogenesis and upstream angiogenic and mechanosensitive pathways., Mechanical cues influence tissue regeneration, and although vasculature is known to be mechanically sensitive, little is known about the effects of bulk extracellular matrix deformation on the nascent vessel networks found in healing tissues. Previously, we found that dynamic matrix compression in vivo potently regulated revascularization during bone tissue regeneration; however, whether matrix deformations directly regulate angiogenesis remained unknown. Here, we demonstrated that load initiation time, magnitude, and mode all regulate microvascular growth, as well as upstream angiogenic and mechanotransduction signaling pathways. Immediate load initiation inhibited angiogenesis and expression of early sprout tip cell selection genes, while delayed loading enhanced microvascular network formation and upstream signaling pathways. This research provides 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.

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Mesenchymal stromal cells (MSCs) have shown promise as a treatment for osteoarthritis (OA); however, effective translation has been limited by numerous factors ranging from high variability and heterogeneity of hMSCs, to suboptimal delivery strategies, to poor understanding of critical quality and potency attributes. The objective of the current study was to assess the effects of biomaterial encapsulation in alginate microcapsules on human MSC (hMSC) secretion of immunomodulatory cytokines in an OA microenvironment and therapeutic efficacy in treating established OA. Lewis rats underwent Medial Meniscal Transection (MMT) surgery to induce OA. Three weeks post-surgery, after OA was established, rats received intra-articular injections of either encapsulated hMSCs or controls (saline, empty capsules, or non-encapsulated hMSCs). Six weeks post-surgery, microstructural changes in the knee joint were quantified using contrast enhanced microCT. Encapsulated hMSCs attenuated progression of OA including articular cartilage degeneration (swelling and cartilage loss) and subchondral bone remodeling (thickening and hardening). A multiplexed immunoassay panel (41 cytokines) was used to profile the in vitro secretome of encapsulated and non-encapsulated hMSCs in response to IL-1□, a key cytokine involved in OA. Non-encapsulated hMSCs showed an indiscriminate increase in all cytokines in response to IL-1□ while encapsulated hMSCs showed a highly targeted secretory response with increased expression of some pro-inflammatory (IL-1β, IL-6, IL-7, IL-8), anti-inflammatory (IL-1RA), and chemotactic (G-CSF, MDC, IP10) cytokines. These data show that biomaterial encapsulation using alginate microcapsules can modulate hMSC paracrine signaling in response to OA cytokines and enhance the therapeutic efficacy of the hMSCs in treating established OA.

Regeneration of skeletal muscle after volumetric injury is thought to be impaired by a dysregulated immune microenvironment that hinders endogenous repair mechanisms. Such defects result in fatty infiltration, tissue scarring, chronic inflammation, and debilitating functional deficits. Here, we evaluated the key cellular processes driving dysregulation in the injury niche through localized modulation of sphingosine-1-phosphate (S1P) receptor signaling. We employ dimensionality reduction and pseudotime analysis on single cell cytometry data to reveal heterogeneous immune cell subsets infiltrating pre-clinical muscle defects due to S1P receptor inhibition. We show that global knockout of S1P receptor 3 (S1PR3) is marked by an increase of muscle stem cells within injured tissue, a reduction in classically-activated relative to alternatively-activated macrophages, and increased bridging of regenerating myofibers across the defect. We found that local S1PR3 antagonism via nanofiber delivery of VPC01091 replicated key features of pseudotime immune cell recruitment dynamics and enhanced regeneration characteristic of global S1PR3 knockout. Our results indicate that local S1P receptor modulation may provide an effective immunotherapy for promoting a pro-reparative environment leading to improved regeneration following muscle injury.

As a potential treatment for osteoarthritis (OA), we have developed injectable and hydrolytically degradable heparin-based biomaterials with tunable sulfation for the intra-articular delivery of tumor necrosis factor-alpha stimulated gene-6 (TSG-6), a protein known to inhibit plasmin which may degrade extracellular matrix within OA joints. We first assessed the effect of heparin sulfation on TSG-6 anti-plasmin activity and found that while fully sulfated (Hep) and heparin desulfated at only the N position (Hep-N) significantly enhanced TSG-6 bioactivity in vitro, fully desulfated heparin (Hep-) had no effect, indicating that heparin sulfation plays a significant role in modulating TSG-6 bioactivity. Next, TSG-6 loaded, degradable 10 wt% Hep-N microparticles (MPs) were delivered via intra-articular injection into the knee at 1, 7, and 15 days following medial meniscal transection (MMT) injury in a rat model. After 21 days, cartilage thickness, volume, and attenuation were significantly increased with soluble TSG-6, indicating degenerative changes. In contrast, no significant differences were observed with TSG-6 loaded MP treatment, demonstrating that TSG-6 loaded MPs reduced cartilage damage following MMT injury. Ultimately, our results indicate that Hep-N can enhance TSG-6 anti-plasmin activity and that Hep-N-based biomaterials may be an effective method for TSG-6 delivery to treat OA.

The growing field of regenerative rehabilitation has great potential to improve clinical outcomes for individuals with disabilities. However, the science to elucidate the specific biological underpinnings of regenerative rehabilitation-based approaches is still in its infancy and critical questions regarding clinical translation and implementation still exist. In a recent roundtable discussion from International Consortium for Regenerative Rehabilitation stakeholders, key challenges to progress in the field were identified. The goal of this article is to summarize those discussions and to initiate a broader discussion among clinicians and scientists across the fields of regenerative medicine and rehabilitation science to ultimately progress regenerative rehabilitation from an emerging field to an established interdisciplinary one. Strategies and case studies from consortium institutions-including interdisciplinary research centers, formalized courses, degree programs, international symposia, and collaborative grants-are presented. We propose that these strategic directions have the potential to engage and train clinical practitioners and basic scientists, transform clinical practice, and, ultimately, optimize patient outcomes.

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.

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.

Type III fractures typically involve segmental bone loss with extensive adjacent soft tissue injury to muscle and vasculature. Such severe composite injuries to bone and muscle often require multiple treatment procedures and are associated with significantly higher rates of complications, including non-union, infection, prolonged disability, and amputation. Successful bone healing depends on early re-vascularization to restore oxygen, nutrient, growth factor, and progenitor cell supply to the injury. Therapeutic angiogenesis strategies have therefore been investigated to promote re-vascularization following severe bone injuries, however results have been inconsistent. In this study, co-stimulation of microvascular fragment constructs with vascular endothelial growth factor (VEGF) and platelet-derived growth factor (PDGF) promoted vascular network formation in vitro compared to VEGF or PDGF alone. In an in vivo model of segmental bone and volumetric muscle loss injury, combined delivery of VEGF and PDGF with a low dose of bone morphogenetic protein-2 (BMP-2) significantly enhanced regeneration of vascularized bone compared to BMP-2 treatment alone. This study demonstrates the potential for a combined osteoinductive and angiogenic growth factor delivery strategy to promote functional bone regeneration following severe composite musculoskeletal injury.

The hierarchical and anisotropic mechanical behavior requirement of load-bearing soft tissues limits the utility of conventional elastomeric materials as a replacement for soft-tissue materials. Liquid-crystal elastomers (LCEs) have the potential to excel in this regard owing to its unique combination of mesogenic order in an elastomeric network. In this study, the mechanical behavior of the LCEs relevant to load-bearing biomedical applications was explored. LCEs with different network orientations (i.e., mesogen alignments) were investigated by fabricating the LCEs with polydomain and monodomain configurations. The polydomain and monodomain LCEs with the same degree of network crosslinking demonstrated diverse mechanical behavior, ranging from highly stiff and elastic nature to high damping capacity, depending on the loading direction with respect to the network alignment. The LCEs were also capable of matching the anisotropic mechanical behavior of an intervertebral disc. Additional studies were conducted on the in vivo biological response of LCEs upon subcutaneous implantation, as well as on the effect of the exposure to an in vitro simulated physiological environment on the mechanical behavior. The LCEs' mechanical response was negligibly affected when exposed to biomedically relevant conditions. Furthermore, the solid and porous LCEs did not show any adverse effect on the surrounding tissues when implanted subcutaneously in rats. The biological response allows for tissue ingrowth and helps illustrate their utility in implantable biological devices. Finally, the utility of LCEs to mimic the mechanical function of biological tissue such as intervertebral disc was demonstrated by fabricating a proof of concept total disc replacement device.

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 longitudinally measured strain across a bone defect in real-time throughout rehabilitation. The results showed that load-sharing permitted by a load-sharing fixator initially delivered a two-fold increase in deformation magnitude, subsequently increased mineralized bridging by nearly three-fold, and increased bone formation by over 60%. These data implicate a critical role for early mechanical cues on the long term healing response as strain cycle magnitude at 1 week (before appreciable healing occurred) had a significant positive correlation with the long-term bone regeneration outcomes. Furthermore, we found that sensor readings correlated with the status of healing, suggesting a role for strain sensing as an X-ray-free healing assessment platform. Therefore, non-invasive strain measurements may possess diagnostic potential to evaluate bone repair and reduce clinical reliance on current radiation-emitting imaging methods. Together, this study demonstrates a promising framework to quantitatively develop and exploit mechanical rehabilitation strategies 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.

Summary Objective The development of effective therapies for cartilage protection has been limited by a lack of efficient quantitative cartilage imaging modalities in pre-clinical in vivo models. Our objectives were two-fold: first, to validate a new contrast-enhanced 3D imaging analysis technique, equilibrium partitioning of an ionic contrast agent-micro computed tomography (EPIC-μCT), in a rat medial meniscal transection (MMT) osteoarthritis (OA) model; and second, to quantitatively assess the sensitivity of EPIC-μCT to detect the effects of matrix metalloproteinase inhibitor (MMPi) therapy on cartilage degeneration. Methods Rats underwent MMT surgery and tissues were harvested at 1, 2, and 3 weeks post-surgery or rats received an MMPi or vehicle treatment and tissues harvested 3 weeks post-surgery. Parameters of disease progression were evaluated using histopathology and EPIC-μCT. Correlations and power analyses were performed to compare the techniques. Results EPIC-μCT was shown to provide simultaneous 3D quantification of multiple parameters, including cartilage degeneration and osteophyte formation. In MMT animals treated with MMPi, OA progression was attenuated, as measured by 3D parameters such as lesion volume and osteophyte size. A post-hoc power analysis showed that 3D parameters for EPIC-μCT were more sensitive than 2D parameters requiring fewer animals to detect a therapeutic effect of MMPi. 2D parameters were comparable between EPIC-μCT and histopathology. Conclusion This study demonstrated that EPIC-μCT has high sensitivity to provide 3D structural and compositional measurements of cartilage and bone in the joint. EPIC-μCT can be used in combination with histology to provide a comprehensive analysis to screen new potential therapies.

Craniofacial bone loss is a complex clinical problem with limited regenerative solutions. Currently, BMP2 is used as a bone-regenerative therapy in adults, but in pediatric cases of bone loss, it is not FDA-approved due to concerns of life-threatening inflammation and cancer. Development of a bone-regenerative therapy for children will transform our ability to reduce the morbidity associated with current autologous bone grafting techniques. We discovered that JAGGED1 (JAG1) induces cranial neural crest (CNC) cell osteoblast commitment during craniofacial intramembranous ossification, suggesting that exogenous JAG1 delivery is a potential craniofacial bone-regenerative approach. In this study, we found that JAG1 delivery using synthetic hydrogels containing O9-1 cells, a CNC cell line, into critical-sized calvarial defects in C57BL/6 mice provided robust bone-regeneration. Since JAG1 signals through canonical (Hes1/Hey1) and non-canonical (JAK2) NOTCH pathways in CNC cells, we used RNAseq to analyze transcriptional pathways activated in CNC cells treated with JAG1±DAPT, a NOTCH-canonical pathway inhibitor. JAG1 upregulated expression of multiple NOTCH canonical pathway genes (Hes1), which were downregulated in the presence of DAPT. JAG1 also induced bone chemokines (Cxcl1), regulators of cytoskeletal organization and cell migration (Rhou), signaling targets (STAT5), promoters of early osteoblast cell proliferation (Prl2c2, Smurf1andEsrra), and, inhibitors of osteoclasts (Id1). In the presence of DAPT, expression levels ofHes1andCxcl1were decreased, whereas,Prl2c2, Smurf1, Esrra, RhouandId1remain elevated, suggesting that JAG1 induces osteoblast proliferation through these non-canonical genes. Pathway analysis of JAG1+DAPT-treated CNC cells revealed significant upregulation of multiple non-canonical pathways, including the cell cycle, tubulin pathway, regulators ofRunx2initiation and phosphorylation of STAT5 pathway. In total, our data show that JAG1 upregulates multiple pathways involved in osteogenesis, independent of the NOTCH canonical pathway. Moreover, our findings suggest that JAG1 delivery using a synthetic hydrogel, is a bone-regenerative approach with powerful translational potential.

Summary Objective This study examined the effects of reduced and elevated weight bearing on post-traumatic osteoarthritis (PTOA) development, locomotor joint kinematics, and degree of voluntary activity in rats following medial meniscal transection (MMT). Design Twenty-one adult rats were subjected to MMT surgery of the left hindlimb and then assigned to one of three groups: (1) regular (i.e., no intervention), (2) hindlimb immobilization, or (3) treadmill running. Sham surgery was performed in four additional rats. Voluntary wheel run time/distance was measured, and 3D hindlimb kinematics were quantified during treadmill locomotion using biplanar radiography. Rats were euthanized 8 weeks after MMT or sham surgery, and the microstructure of the tibial cartilage and subchondral bone was quantified using contrast enhanced micro-CT. Results All three MMT groups showed signs of PTOA (full-thickness lesions and/or increased cartilage volume) compared to the sham group, however the regular and treadmill-running groups had greater osteophyte formation than the immobilization group. For the immobilization group, increased volume was only observed in the anterior region of the cartilage. The treadmill-running group demonstrated a greater knee varus angle at mid-stance than the sham group, while the immobilization group demonstrated greater reduction in voluntary running than all the other groups at 2 weeks post-surgery. Conclusions Elevated weight-bearing via treadmill running at a slow/moderate speed did not accelerate PTOA in MMT rats when compared to regular weight-bearing. Reduced weight-bearing via immobilization may attenuate overall PTOA but still resulted in regional cartilage degeneration. Overall, there were minimal differences in hindlimb kinematics and voluntary running between MMT and sham rats.

Dysfunctional endothelium causes more disease than any other cell type. Systemically administered RNA delivery to nonliver tissues remains challenging, in large part because there is no high-throughput method to identify nanoparticles that deliver functional mRNA to cells in vivo. Here we report a system capable of simultaneously quantifying how >100 lipid nanoparticles (LNPs) deliver mRNA that is translated into functional protein. Using this system (named FIND), we measured how >250 LNPs delivered mRNA to multiple cell types in vivo and identified 7C2 and 7C3, two LNPs that efficiently deliver siRNA, single-guide RNA (sgRNA), and mRNA to endothelial cells. The 7C3 delivered Cas9 mRNA and sgRNA to splenic endothelial cells as efficiently as hepatocytes, distinguishing it from LNPs that deliver Cas9 mRNA and sgRNA to hepatocytes more than other cell types. These data demonstrate that FIND can identify nanoparticles with novel tropisms in vivo.

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 did not improve healing outcomes, we observed a significant BMP-2 dose-dependent increase in regenerated bone volume and biomechanical properties. This is the first known report of an increased BMP-2 dose improving bone healing with concomitant muscle damage. While high dose BMP-2 delivery can induce heterotopic ossification (HO) and increased inflammation, the maximum 10 μg 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 μg) 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. Published 2019. This article is a U.S. Government work and is in the public domain in the USA. J Orthop Res.

Traumatic musculoskeletal injuries that result in bone defects or fractures often affect both bone and the surrounding soft tissue. Clinically, these types of multi-tissue injuries have increased rates of complications and long-term disability. Vascular integrity is a key clinical indicator of injury severity, and revascularization of the injury site is a critical early step of the bone healing process. Our lab has previously established a pre-clinical model of composite bone-muscle injury that exhibits impaired bone healing; however, the vascularization response in this model had not yet been investigated. Here, the early revascularization of a bone defect following composite injury is shown to be impaired, and subsequently the therapeutic potential of combined vascularization and osteoinduction was investigated to overcome the impaired regeneration in composite injuries. A decorin (DCN)-supplemented collagen hydrogel was developed as a biomaterial delivery vehicle for the co-delivery microvascular fragments (MVF), which are multicellular segments of mature vasculature, and bone morphogenetic protein-2 (BMP-2), a potent osteoinductive growth factor. We hypothesized that collagen + DCN would increase BMP-2 retention over collagen alone due to DCN’s ability to sequester TGF-s growth factors. We further hypothesized that MVF would increase both early vascularization and subsequent BMP-2-mediated bone regeneration. Contrary to our hypothesis, BMP + MVF decreased the number of blood vessels relative to BMP alone and had no effect on bone healing. However, collagen + DCN was demonstrated to be a BMP-2 delivery vehicle capable of achieving bridging in the challenging composite defect model that is comparable to that achieved with a well-established alginate-based delivery system. Statement of Significance We have previously established a model of musculoskeletal trauma that exhibits impaired bone healing. For the first time, this work shows that the early revascularization response is also significantly, albeit modestly, impaired. A decorin-supplemented collagen hydrogel was used for the first time in vivo as a delivery vehicle for both a cell-based vascular therapeutic, MVF, and an osteoinductive growth factor, BMP-2. While MVF did not improve vascular volume or bone healing, collagen + DCN is a BMP-2 delivery vehicle capable of achieving bridging in the challenging composite defect model. Based on its support of robust angiogenesis in vitro, collagen + DCN may be extended for future use with other vascular therapeutics such as pre-formed vascular networks.

During craniofacial development, cranial neural crest (CNC) cells migrate into the developing face and form bone through intramembranous ossification. Loss of JAGGED1 (JAG1) signaling in the CNC cells is associated with maxillary hypoplasia or maxillary bone deficiency (MBD) in mice and recapitulates the MBD seen in humans with Alagille syndrome. JAGGED1, a membrane-bound NOTCH ligand, is required for normal craniofacial development, andJagged1mutations in humans are known to cause Alagille Syndrome, which is associated with cardiac, biliary, and bone phenotypes and these children experience increased bony fractures. Previously, we demonstrated deficient maxillary osteogenesis inWnt1-cre;Jagged1f/f(Jag1CKO) mice by conditional deletion ofJagged1in maxillary CNC cells. In this study, we investigated the JAG1 signaling pathways in a CNC cell line. Treatment with JAG1 induced osteoblast differentiation and maturation markers,Runx2andOcn, respectively, Alkaline Phosphatase (ALP) production, as well as classic NOTCH1 targets,Hes1andHey1.While JAG1-inducedHes1andHey1expression levels were predictably decreased after DAPT (NOTCH inhibitor) treatment, JAG1-inducedRunx2andOcnlevels were surprisingly constant in the presence of DAPT, indicating that JAG1 effects in the CNC cells are independent of the canonical NOTCH pathway. JAG1 treatment of CNC cells increased Janus Kinase 2 (JAK2) phosphorylation, which was refractory to DAPT treatment, highlighting the importance of the non-canonical NOTCH pathway during CNC cells osteoblast commitment. Pharmacologic inhibition of JAK2 phosphorylation, with and without DAPT treatment, upon JAG1 induction reduced ALP production and,Runx2andOcngene expression. Collectively, these data suggest that JAK2 is an essential component downstream of a non-canonical JAG1-NOTCH1 pathway through which JAG1 stimulates expression of osteoblast-specific gene targets in CNC cells that contribute to osteoblast differentiation and bone mineralization.

Skeletal muscle has a remarkable regenerative capacity; however, after volumetric muscle loss (VML) due to traumatic injury or surgery this regenerative response is significantly diminished, causing chronic functional deficits. The critical defect size at which the muscle will not functionally recover has not yet been established and subsequently, the relative contribution of crucial muscle components, including muscle stem cells and the muscle stem cell niche, are unknown. In this study, we created VML injuries of 2, 3, or 4 mm diameter, full-thickness defects in the mouse quadriceps. The 2, 3, and 4 mm injuries resulted in a defect of 5, 15, or 30% of the quadriceps mass, respectively. At 14 and 28 days after injury, histological analyses revealed injury size-dependent differences in myofiber morphology and fibrosis; the number of small myofibers increased with increasing injury size. The results showed that the 3 mm injury was at a threshold point, as myofibers were unable to bridge the defect, there was persistent fibrosis and inflammation, and significantly increased number of myofibers with centrally located nuclei. We then further investigated the 3 mm VML for nerve and vascular regeneration. These injured muscles were accompanied by a drastic increase in denervated neuromuscular junctions (NMJ), while assessment of angiogenesis via micro-CT analysis revealed a significant increase in vascular volume primarily from small diameter vessels after VML injury. Collectively, these data indicate that the spatial and temporal control of the fibrotic and neuromotor response are critical to regeneration and could be potential therapeutic targets, as they are the most dysregulated components of the muscle stem cell niche after VML.