A Mechanistic and Preclinical Assessment of BioRestore Bioactive Glass as a Synthetic Bone Graft Extender and Substitute for Osteoinduction and Spine Fusion.

STUDY DESIGN: Preclinical animal study. OBJECTIVE: Evaluate the osteoinductivity and bone regenerative capacity of BioRestore bioactive glass. SUMMARY OF BACKGROUND DATA: BioRestore is a Food and Drug Administration (FDA)-approved bone void filler that has not yet been evaluated as a bone graft extender or substitute for spine fusion. METHODS: In vitro and in vivo methods were used to compare BioRestore with other biomaterials for the capacity to promote osteodifferentiation and spinal fusion. The materials evaluated (1) absorbable collagen sponge (ACS), (2) allograft, (3) BioRestore, (4) Human Demineralized Bone Matrix (DBM), and (5) MasterGraft. For in vitro studies, rat bone marrow-derived stem cells (BMSC) were cultured on the materials in either standard or osteogenic media (SM, OM), followed by quantification of osteogenic marker genes (Runx2, Osx, Alpl, Bglap, Spp1) and alkaline phosphatase (ALP) activity. Sixty female Fischer rats underwent L4-5 posterolateral fusion (PLF) with placement of 1 of 5 implants: (1) ICBG from syngeneic rats; (2) ICBG+BioRestore; (3) BioRestore alone; (4) ICBG+Allograft; or (5) ICBG+MasterGraft. Spines were harvested 8 weeks postoperatively and evaluated for bone formation and fusion via radiography, blinded manual palpation, microCT, and histology. RESULTS: After culture for 1 week, BioRestore promoted similar expression levels of Runx2 and Osx to cells grown on DBM. At the 2-week timepoint, the relative ALP activity for BioRestore-OM was significantly higher (P<0.001) than that of ACS-OM and DBM-OM (P<0.01) and statistically equivalent to cells grown on allograft-OM. In vivo, radiographic and microCT evaluation showed some degree of bridging bone formation in all groups tested, with the exception of BioRestore alone, which did not produce successful fusions. CONCLUSIONS: This study demonstrates the capacity of BioRestore to promote osteoinductivity in vitro. In vivo, BioRestore performed similarly to commercially available bone graft extender materials but was incapable of producing fusion as a bone graft substitute. LEVEL OF EVIDENCE: Level V.

Sex-based Difference in Response to Recombinant Human Bone Morphogenetic Protein-2 in a Rat Posterolateral Fusion Model.

STUDY DESIGN: This was a preclinical study. OBJECTIVE: Evaluate sex-dependent differences in the bone healing response to recombinant human bone morphogenetic protein-2 (rhBMP-2) in a rat posterolateral spinal fusion model. SUMMARY OF BACKGROUND DATA: Minimal and conflicting data exist concerning potential sex-dependent differences in rhBMP-2-mediated bone regeneration in the context of spinal fusion. MATERIALS AND METHODS: Forty-eight female and male Sprague-Dawley rats (N=24/group), underwent L4-L5 posterolateral fusion with bilateral placement of an absorbable collagen sponge, each loaded with 5 µg of bone morphogenetic protein-2 (10 µg/animal). At eight weeks postoperative, 10 specimens of each sex were tested in flexion-extension with quantification of range of motion and stiffness. The remaining specimens were evaluated for new bone growth and successful fusion via radiography, blinded manual palpation and microcomputed tomography (microCT). Laboratory microCT quantified bone microarchitecture, and synchrotron microCT examined bone microstructure at the 1 µm level. RESULTS: Manual palpation scores differed significantly between sexes, with mean fusion scores of 2.4±0.4 in females versus 3.1±0.6 in males ( P <0.001). Biomechanical stiffness did not differ between sexes, but range of motion was significantly greater and more variable for females versus males (3.7±5.6° vs. 0.27±0.15°, P <0.005, respectively). Laboratory microCT showed significantly smaller volumes of fusion masses in females versus males (262±87 vs. 732±238 mm 3 , respectively, P <0.001) but significantly higher bone volume fraction (0.27±0.08 vs. 0.12±0.05, respectively, P <0.001). Mean trabecular thickness was not different, but trabecular number was significantly greater in females (3.1±0.5 vs. 1.5±0.4 mm -1 , respectively, P <0.001). Synchrotron microCT showed fine bone structures developing in both sexes at the eight-week time point. CONCLUSIONS: This study demonstrates sex-dependent differences in bone regeneration induced by rhBMP-2. Further investigation is needed to uncover the extent of and mechanisms underlying these sex differences, particularly at different doses of rhBMP-2.

Preclinical Safety of a 3D-Printed Hydroxyapatite-Demineralized Bone Matrix Scaffold for Spinal Fusion.

STUDY DESIGN: Prospective, randomized, controlled preclinical study. OBJECTIVE: The objective of this study was to compare the host inflammatory response of our previously described hyperelastic, 3D-printed (3DP) hydroxyapatite (HA)-demineralized bone matrix (DBM) composite scaffold to the response elicited with the use of recombinant human bone morphogenetic protein-2 (rhBMP-2) in a preclinical rat posterolateral lumbar fusion model. SUMMARY OF BACKGROUND DATA: Our group previously found that this 3D-printed HA-DBM composite material shows promise as a bone graft substitute in a preclinical rodent model, but its safety profile had yet to be assessed. METHODS: Sixty female Sprague-Dawley rats underwent bilateral posterolateral intertransverse lumbar spinal fusion using with the following implants: 1) type I absorbable collagen sponge (ACS) alone; 2) 10 µg rhBMP-2/ACS; or 3) the 3DP HA-DBM composite scaffold (n = 20). The host inflammatory response was assessed using magnetic resonance imaging, while the local and circulating cytokine expression levels were evaluated by enzyme-linked immunosorbent assays at subsequent postoperative time points (N = 5/time point). RESULTS: At both 2 and 5 days postoperatively, treatment with the HA-DBM scaffold produced significantly less soft tissue edema at the fusion bed site relative to rhBMP-2-treated animals as quantified on magnetic resonance imaging. At every postoperative time point evaluated, the level of soft tissue edema in HA-DBM-treated animals was comparable to that of the ACS control group. At 2 days postoperatively, serum concentrations of tumor necrosis factor-α and macrophage chemoattractant protein-1 were significantly elevated in the rhBMP-2 treatment group relative to ACS controls, whereas these cytokines were not elevated in the HA-DBM-treated animals. CONCLUSION: The 3D-printed HA-DBM composite induces a significantly reduced host inflammatory response in a preclinical spinal fusion model relative to rhBMP-2.Level of Evidence: N/A.

Improved Posterolateral Lumbar Spinal Fusion Using a Biomimetic, Nanocomposite Scaffold Augmented by Autologous Platelet-Rich Plasma.

Remodeling of the human bony skeleton is constantly occurring with up to 10% annual bone volume turnover from osteoclastic and osteoblastic activity. A shift toward resorption can result in osteoporosis and pathologic fractures, while a shift toward deposition is required after traumatic, or surgical injury. Spinal fusion represents one such state, requiring a substantial regenerative response to immobilize adjacent vertebrae through bony union. Autologous bone grafts were used extensively prior to the advent of advanced therapeutics incorporating exogenous growth factors and biomaterials. Besides cost constraints, these applications have demonstrated patient safety concerns. This study evaluated the regenerative ability of a nanostructured, magnesium-doped, hydroxyapatite/type I collagen scaffold (MHA/Coll) augmented by autologous platelet-rich plasma (PRP) in an orthotopic model of posterolateral lumbar spinal fusion. After bilateral decortication, rabbits received either the scaffold alone (Group 1) or scaffold with PRP (Group 2) to the anatomic right side. Bone regeneration and fusion success compared to internal control were assessed by DynaCT with 3-D reconstruction at 2, 4, and 6 weeks postoperatively followed by comparative osteogenic gene expression and representative histopathology. Both groups formed significantly more new bone volume than control, and Group 2 subjects produced significantly more trabecular and cortical bone than Group 1 subjects. Successful fusion was seen in one Group 1 animal (12.5%) and 6/8 Group 2 animals (75%). This enhanced effect by autologous PRP treatment appears to occur via astounding upregulation of key osteogenic genes. Both groups demonstrated significant gene upregulation compared to vertebral bone controls for all genes. Group 1 averaged 2.21-fold upregulation of RUNX2 gene, 3.20-fold upregulation of SPARC gene, and 3.67-fold upregulation of SPP1 gene. Depending on anatomical subgroup (cranial, mid, caudal scaffold portions), Group 2 had significantly higher average expression of all genes than both control and Group 1-RUNX2 (8.23-19.74 fold), SPARC (18.67-55.44 fold), and SPP1 (46.09-90.65 fold). Our data collectively demonstrate the osteoinductive nature of a nanostructured MHA/Coll scaffold, a beneficial effect of augmentation with autologous PRP, and an ability to achieve clinical fusion when applied together in an orthotopic model. This has implications both for future study and biomedical innovation of bone-forming therapeutics.

Osteoinductivity and biomechanical assessment of a 3D printed demineralized bone matrix-ceramic composite in a rat spine fusion model.

We recently developed a recombinant growth factor-free bone regenerative scaffold composed of stoichiometric hydroxyapatite (HA) ceramic particles and human demineralized bone matrix (DBM) particles (HA-DBM). Here, we performed the first pre-clinical comparative evaluation of HA-DBM relative to the industry standard and established positive control, recombinant human bone morphogenetic protein-2 (rhBMP-2), using a rat posterolateral spinal fusion model (PLF). Female Sprague-Dawley rats underwent bilateral L4-L5 PLF with implantation of the HA-DBM scaffold or rhBMP-2. Fusion was evaluated using radiography and blinded manual palpation, while biomechanical testing quantified the segmental flexion-extension range-of-motion (ROM) and stiffness of the fused segments at 8-weeks postoperatively. For mechanistic studies, pro-osteogenic gene and protein expression at 2-days and 1-, 2-, and 8-weeks postoperatively was assessed with another cohort. Unilateral fusion rates did not differ between the HA-DBM (93%) and rhBMP-2 (100%) groups; however, fusion scores were higher with rhBMP-2 (p = 0.008). Both treatments resulted in significantly reduced segmental ROM (p < 0.001) and greater stiffness (p = 0.009) when compared with non-operated controls; however, the degree of stabilization was significantly higher with rhBMP-2 treatment relative to the HA-DBM scaffold. In the mechanistic studies, PLGA and HA scaffolds were used as negative controls. Both rhBMP-2 and HA-DBM treatments resulted in significant elevations of several osteogenesis-associated genes, including Runx2, Osx, and Alp. The rhBMP-2 treatment led to significantly greater early, mid, and late osteogenic markers, which may be the mechanism in which early clinical complications are seen. The HA-DBM scaffold also induced osteogenic gene expression, but primarily at the 2-week postoperative timepoint. Overall, our findings show promise for this 3D-printed composite as a recombinant growth factor-free bone graft substitute for spinal fusion. STATEMENT OF SIGNIFICANCE: Despite current developments in bone graft technology, there remains a significant void in adequate materials for bone regeneration in clinical applications. Two of the most efficacious bone graft options are the gold-standard iliac crest bone graft and recombinant human-derived bone morphogenetic protein-2 (rhBMP-2), available commercially as Infuse™. Although efficacious, autologous graft is associated with donor-site morbidity, and Infuse™ has known side effects related to its substantial host inflammatory response, possibly associated with a immediate, robust osteoinductive response. Hence, there is a need for a bone graft substitute that provides adequate osteogenesis without associated adverse events. This study represents a significant step in the design of off-the-shelf growth factor-free devices for spine fusion.

Characterizing the host response to rhPDGF-BB in a rat spinal arthrodesis model.

BACKGROUND: Due to the constraints surrounding autograft bone, surgeons have turned to osteoinductive agents to augment spinal fusion. Reports of complications and questionable efficacy slowed the adoption of these alternatives. Recombinant human platelet-derived growth factor B homodimer (rhPDGF-BB) has been Food and Drug Administration (FDA)-approved (Augment) to promote fusion in other areas of orthopedics, but its characterization in spine fusion has not yet been tested. The purpose of this study is to characterize the host response to PDGF-BB in vivo. METHODS: Eighty female Fischer rats underwent L4-5 posterolateral fusion using one of four implant types: (a) iliac crest syngeneic allograft harvested from syngeneic donors, (b) ß-TCP/bovine collagen matrix (ß-TCP/Col) with sodium acetate buffer, (c) ß-TCP/Col with 0.3 mg/mL "low dose," or (d) ß-TCP/Col with 3.0 mg/mL "high dose" of rhPDGF-BB. Animals underwent magnetic resonance imaging (MRI) and serum cytokine quantification at 4, 7, 10, and 21 days, postoperatively. Tissues were processed for immunofluorescence staining for Ki67 and von Willebrand factor (vWF) to assess neovascularization. RESULTS: MRI demonstrated no differences in fluid accumulation among the four treatment groups at any of the time points. Serum cytokine analysis showed no clinically significant differences between treatment groups in 20 of the 27 cytokines. Inflammatory cytokines IFN-γ, IL-1ß, IL-18, MCP-1, MIP-1α, TNF-α were not induced by rhPDGF-BB. Histology showed no differences in cell infiltration, and Ki67 and vWF immunofluorescence staining was similar among groups. CONCLUSIONS: rhPDGF-BB delivered with a ß-TCP/Col matrix exerts no exaggerated systemic or local host inflammatory response when compared to iliac crest syngeneic allograft bone or the control carrier. rhPDGF-BB mixed with a ß-TCP/Col matrix could be a viable and safe biologic alternative to syngeneic allograft in spine fusion. Further studies need to be performed to evaluate efficacy in this setting.

The effect of local steroid application on bony fusion in a rat posterolateral spinal arthrodesis model.

INTRODUCTION: Local steroid administration during anterior cervical spine surgery has been shown to improve postoperative dysphagia. However, concerns over potential complications remain. This study aims to evaluate the effect of local steroid administration on bone regeneration and spine fusion in a preclinical model, as well as the impact on osteogenic differentiation of human bone marrow-derived mesenchymal stem cells (hBM-MSCs) in a 3D culture system. MATERIALS AND METHODS: Forty-five rats underwent bilateral L4-L5 posterolateral lumbar fusion (PLF) utilizing local delivery of low-dose recombinant human bone morphogenetic protein-2 (rhBMP-2; 0.5 µg/implant). Rats were divided into three groups: no steroid (control), low dose (0.5 mg/kg), and high dose (2.5 mg/kg) of triamcinolone. Bone growth and fusion were assessed using radiography, blinded manual palpation, and micro-CT analysis and were visualized by histology. The impact of triamcinolone exposure on osteogenic differentiation of hBM-MSCs was evaluated by gene expression analysis, alkaline phosphatase activity assay, and alizarin red staining. RESULTS: No significant differences in fusion scores or rates were seen in the low- or high-dose steroid treatment groups relative to untreated controls. Quantification of new bone formation via micro-CT imaging revealed no significant between-group differences in the volume of newly regenerated bone. Triamcinolone also had no negative impact on pro-osteogenic gene transcript levels, and ALP activity was enhanced in the presence of triamcinolone. Mineral deposition appeared comparable in cultures grown with and without triamcinolone. CONCLUSIONS: Local steroid application does not seem to inhibit rhBMP-2-mediated spine fusion in rats, though our study may not be adequately powered to detect differences in fusion as measured by manual palpation or bone volume as measured by micro-CT. These findings suggest that local triamcinolone may not increase pseudarthrosis in spine fusion procedures. Further large animal and clinical studies to verify its safety and efficacy are warranted.

Nanostructured Biomaterials for Bone Regeneration.

This review article addresses the various aspects of nano-biomaterials used in or being pursued for the purpose of promoting bone regeneration. In the last decade, significant growth in the fields of polymer sciences, nanotechnology, and biotechnology has resulted in the development of new nano-biomaterials. These are extensively explored as drug delivery carriers and as implantable devices. At the interface of nanomaterials and biological systems, the organic and synthetic worlds have merged over the past two decades, forming a new scientific field incorporating nano-material design for biological applications. For this field to evolve, there is a need to understand the dynamic forces and molecular components that shape these interactions and influence function, while also considering safety. While there is still much to learn about the bio-physicochemical interactions at the interface, we are at a point where pockets of accumulated knowledge can provide a conceptual framework to guide further exploration and inform future product development. This review is intended as a resource for academics, scientists, and physicians working in the field of orthopedics and bone repair.

Bioinspired Scaffold Action Under the Extreme Physiological Conditions of Simulated Space Flights: Osteogenesis Enhancing Under Microgravity.

Prolonged exposure to microgravity (MG) during long-duration space flights is known to induce severe dysregulation of osteoblast functions connected to a significant bone loss, similar to the condition induced by osteoporosis. Hence, we here present MG as a promising model to challenge the effectiveness of new scaffolds designed for bone regeneration in counteracting bone loss. To this end, we carried out an integrative study aimed to evaluate, in the extreme condition of Random Positioning Machine-simulated MG, the osteoinductive potential of nanocrystalline magnesium-doped hydroxyapatite/type I collagen composite scaffold (MHA/Coll), that we previously demonstrated to be an excellent tool for bone tissue engineering. Initially, to test the osteoinductive properties of our bioinspired-scaffold, MHA/Coll structure was fully characterized under MG condition and compared to its static counterpart. Human bone marrow-derived mesenchymal stem cells were used to investigate the scaffold biocompatibility and ability to promote osteogenic differentiation after long-duration exposure to MG (up to 21 days). The results demonstrate that the nanostructure of MHA/Coll scaffold can alleviate MG-induced osteoblast dysfunction, promoting cell differentiation along the osteogenic lineage, with a consequent reduction in the expression of the surface markers CD29, CD44, and CD90. Moreover, these findings were corroborated by the ability of MHA/Coll to induce the expression of genes linked to osteogenesis, including alkaline phosphatase and osteocalcin. This study confirmed MHA/Coll capabilities in promoting osteogenesis even in extreme long-term condition of MG, suggesting MG as an effective challenging model to apply in future studies to validate the ability of advanced scaffolds to counteract bone loss, facilitating their application in translational Regenerative Medicine and Tissue Engineering.

Mimicking the Organic and Inorganic Composition of Anabolic Bone Enhances Human Mesenchymal Stem Cell Osteoinduction and Scaffold Mechanical Properties.

Engineered bone graft designs have been largely inspired by adult bone despite functionally significant differences from the composition of anabolic bone in both the mineralized and non-mineralized fractions. Specifically, anabolic bone contains hydroxyapatite with ionic substitutions that facilitate bone turnover and relatively rare collagens type VI and XII that are important for normal bone development. In this work, human mesenchymal stem cells (hMSCs) were cultured in lyophilized collagen type I scaffolds mineralized with hydroxyapatite containing Mg2+ substitutions, then induced to deposit an extracellular matrix (ECM) containing collagens VI and XII by exposure to GW9662, a PPARγ inhibitor. Delivery of GW9662 was accomplished through either Supplemented Media or via composite microspheres embedded in the scaffolds for localized delivery. Furthermore, hMSCs and scaffolds were cultured in both static and perfuse conditions to investigate the interaction between GW9662 treatment and perfusion and their effects on ECM deposition trends. Perfusion culture enhanced cell infiltration into the scaffold, deposition of collagen VI and XII, as well as osteogenic differentiation, as determined by gene expression of osteopontin, BMP2, and ALP. Furthermore, scaffold mineral density and compressive modulus were increased in response to both GW9662 treatment and perfusion after 3 weeks of culture. Local delivery of GW9662 with drug-eluting microspheres had comparable effects to systemic delivery in the perfusate. Together, these results demonstrate a strategy to create a scaffold mimicking both organic and inorganic characteristics of anabolic bone and its potential as a bone graft.

Combined continuous glucose monitoring and subcutaneous insulin infusion versus self-monitoring of blood glucose with optimized multiple injections in people with type 1 diabetes: A randomized crossover trial.

AIM: To investigate the efficacy of a combination of continuous glucose monitoring (CGM) and continuous subcutaneous insulin infusion (CSII) versus an optimized degludec-based multiple daily injections (MDI) regimen + self-monitoring of blood glucose (SMBG) in people with type 1 diabetes with regard to optimizing glucose control. MATERIAL AND METHODS: The trial included 28 individuals who underwent a 4-week run-in phase, and were then randomized 1:1 to: (a) CSII + CGM followed by MDI + SMBG or (b) an MDI basal-bolus regimen followed by CSII + CGM. RESULTS: In patients randomized to the CSII + CGM → MDI + SMBG arm, a significant reduction in glycated haemoglobin (HbA1c) versus baseline was found at the end of the first phase (CSII + CGM) without significant variation in the following MDI + SMBG phase. In the arm randomized to the MDI + SMBG → CSII + CGM sequence, a significant improvement in HbA1c was observed in the first phase (MDI + SMBG), together with a further decrease in the following CSII + CGM phase. In the comparison of the two treatments using a mixed linear model, CSII + CGM was superior to MDI + SMBG with respect to change in HbA1c (P = 0.001). CONCLUSIONS: This study suggests that CSII + CGM improves glycaemic control without relevant safety issues in type 1 diabetes, in comparison with MDI + SMBG.

Biocompatible PLGA-Mesoporous Silicon Microspheres for the Controlled Release of BMP-2 for Bone Augmentation.

Bone morphogenetic protein-2 (BMP-2) has been demonstrated to be one of the most vital osteogenic factors for bone augmentation. However, its uncontrolled administration has been associated with catastrophic side effects, which compromised its clinical use. To overcome these limitations, we aimed at developing a safer controlled and sustained release of BMP-2, utilizing poly(lactic-co-glycolic acid)-multistage vector composite microspheres (PLGA-MSV). The loading and release of BMP-2 from PLGA-MSV and its osteogenic potential in vitro and in vivo was evaluated. BMP-2 in vitro release kinetics was assessed by ELISA assay. It was found that PLGA-MSV achieved a longer and sustained release of BMP-2. Cell cytotoxicity and differentiation were evaluated in vitro by MTT and alkaline phosphatase (ALP) activity assays, respectively, with rat mesenchymal stem cells. The MTT results confirmed that PLGA-MSVs were not toxic to cells. ALP test demonstrated that the bioactivity of BMP-2 released from the PLGA-MSV was preserved, as it allowed for the osteogenic differentiation of rat mesenchymal stem cells, in vitro. The biocompatible, biodegradable, and osteogenic PLGA-MSVs system could be an ideal candidate for the safe use of BMP-2 in orthopedic tissue engineering applications.

An elastin-based vasculogenic scaffold promotes marginal islet mass engraftment and function at an extrahepatic site.

In islet transplantation, one of the major obstacles to optimal engraftment is the loss of islet natural vascularization and islet-specific extracellular matrix (ECM) during the islet isolation process. Thus, transplanted islets must re-establish nutritional and physical support through formation of new blood vessels and new ECM. To promote this critical process, we developed an elastin-based vasculogenic and ECM-promoting scaffold engineered for extrahepatic islet transplantation. The scaffold by design consisted of type I collagen (Coll) blended with 20wt% of elastin (E) shown to promote angiogenesis as well as de novo ECM deposition. The resulting "CollE" scaffolds h ad interconnected pores with a size distribution tailored to accommodate seeding of islets as well as growth of new blood vessels. In vitro, CollE scaffolds enabled prolonged culture of murine islets for up to one week while preserving their integrity, viability and function. In vivo, after only four weeks post-transplant of a marginal islet mass, CollE scaffolds demonstrated enhanced vascularization of the transplanted islets in the epididymal fat pad and promoted a prompt reversal of hyperglycemia in previously diabetic recipients. This outcome was comparable to that of kidney capsular (KC) islet transplantation, and superior to that of islets transplanted on the control collagen-only scaffolds (Coll). Crucial genes associated with angiogenesis (VEGFA, PDGFB, FGF1, and COL3A1) as well as de novo islet-specific matrix deposition (COL6A1, COL4A1, LAMA2 and FN1) were all significantly upregulated in islets on CollE scaffolds in comparison to those on Coll scaffolds. Finally, CollE scaffolds were also able to support human islet culture in vitro. In conclusion, CollE scaffolds have the potential to improve the clinical outcome of marginal islet transplantation at extrahepatic sites by promoting angiogenesis and islet-specific ECM deposition.

A biomimetic 3D model of hypoxia-driven cancer progression.

The fate of tumors depends both on the cancer cells' intrinsic characteristics and on the environmental conditions where the tumors reside and grow. Engineered in vitro models have led to significant advances in cancer research, allowing the investigation of cells in physiological environments and the study of disease mechanisms and processes with enhanced relevance. Here we present a biomimetic cancer model based on a collagen matrix synthesized through a biologically inspired process. We compared in this environment the responses of two breast tumor lineages characterized by different molecular patterns and opposite clinical behaviors: MCF-7 that belong to the luminal A subtype connected to an indolent course, and basal-like MDA-MB-231 connected to high-grade and aggressive disease. Cancer cells in the biomimetic matrix recreate a hypoxic environment that affects their growth dynamics and phenotypic features. Hypoxia induces apoptosis and the selection of aggressive cells that acquire expression signatures associated with glycolysis, angiogenesis, cell-matrix interaction, epithelial to mesenchymal transition and metastatic ability. In response to hypoxia MDA-MB-231 migrate on the collagen fibrils and undergo cellular senescence, while MCF-7 do not exhibit these behaviors. Our biomimetic model mimics the evolution of tumors with different grade of aggressiveness fostered by a hypoxic niche and provides a relevant technology to dissect the events involved in cancer progression.

Electrospun Patch Functionalized with Nanoparticles Allows for Spatiotemporal Release of VEGF and PDGF-BB Promoting In Vivo Neovascularization.

The use of nanomaterials as carriers for the delivery of growth factors has been applied to a multitude of applications in tissue engineering. However, issues of toxicity, stability, and systemic effects of these platforms have yet to be fully understood, especially for cardiovascular applications. Here, we proposed a delivery system composed of poly(dl-lactide- co-glycolide) acid (PLGA) and porous silica nanoparticles (pSi) to deliver vascular endothelial growth factor (VEGF) and platelet-derived growth factor (PDGF). The tight spatiotemporal release of these two proteins has been proven to promote neovascularization. In order to minimize tissue toxicity, localize the release, and maintain a stable platform, we conjugated two formulations of PLGA-pSi to electrospun (ES) gelatin to create a combined ES patch releasing both PDGF and VEGF. When compared to freely dispersed particles, the ES patch cultured in vitro with neonatal cardiac cells had significantly less particle internalization (2.0 ± 1.3%) compared to free PLGA-pSi (21.5 ± 6.1) or pSi (28.7 ± 2.5) groups. Internalization was positively correlated to late-stage apoptosis with PLGA-pSi and pSi groups having increased apoptosis compared to the untreated group. When implanted subcutaneously, the ES patch was shown to have greater neovascularization than controls evidenced by increased expression of α-SMA and CD31 after 21 days. Quantitative reverse transcription-polymerase chain reaction results support increased angiogenesis by the upregulation of VEGFA, VEGFR2, vWF, and COL3A1, exhibiting a synergistic effect with the release of VEGF-A164 and PDGF-BB after 21 days in vivo. The results of this study proved that the ES patch reduced cellular toxicity and may be tailored to have a dual release of growth factors promoting localized neovascularization.

Biomimetic nanoparticles for transplantation tolerance.

PURPOSE OF REVIEW: The current review aims to provide a current landscape and future trends of biomimetic nanoparticles which have the potential to revolutionize the field of transplantation in the next decade. RECENT FINDINGS: Currently, the inability to safely induce robust donor-specific immunological tolerance makes it difficult to achieve immunosuppression-free graft survival. Despite progresses in the development of nanotherapeutics for antigen-specific immunomodulation in autoimmune diseases and in cancer treatments, few have been proposed and tested in transplantation with success. The complexity of parallel rejection mechanisms, multitude of antigen epitopes, and potential epitope spreading have challenged conventional nanodelivery systems in transplant models. Overcoming such challenges, biomimetic nanotherapeutics represent a promising alternative, as they allow better recapitulation of the complexity of the main biological players involved in tolerance. Within biomimetic nanodelivery systems, we envision that hybrid systems mimicking extracellular vesicles have the potential to bridging the gap between cell-based therapies, which are effective but costly and difficult to translate in clinical practice, and fully synthetic systems which are relatively easy to manufacture but lack the capacity to recapitulate the complexity of transplant antigens and tolerance mechanisms. SUMMARY: Next-generation nanotherapeutics for tolerance delivery is evolving toward biomimetic systems capable of capturing an increasing level of antigen complexity and exploiting multiple tolerance pathways.

Immune tuning scaffold for the local induction of a pro-regenerative environment.

In mammals, tissue regeneration is accomplished through a well-regulated, complex cascade of events. The disruption of the cellular and molecular processes involved in tissue healing might lead to scar formation. Most tissue engineering approaches have tried to improve the regenerative outcome following an injury, through the combination of biocompatible materials, stem cells and bioactive factors. However, implanted materials can cause further healing impairments due to the persistent inflammatory stimuli that trigger the onset of chronic inflammation. Here, it is described at the molecular, cellular and tissue level, the body response to a functionalized biomimetic collagen scaffold. The grafting of chondroitin sulfate on the surface of the scaffold is able to induce a pro-regenerative environment at the site of a subcutaneous implant. The early in situ recruitment, and sustained local retention of anti-inflammatory macrophages significantly reduced the pro-inflammatory environment and triggered a different healing cascade, ultimately leading to collagen fibril re-organization, blood vessel formation, and scaffold integration with the surrounding native tissue.

Hyaluronic acid coatings as a simple and efficient approach to improve MSC homing toward the site of inflammation.

A major challenge in regenerative medicine is to improve therapeutic cells' delivery and targeting using an efficient and simple protocol. Mesenchymal stem cells (MSC) are currently employed for the treatment of inflammatory-based diseases, due to their powerful immunosoppressive potential. Here we report a simple and versatile method to transiently overexpress the hyaluronic acid (HA) receptor, CD44, on MSC membranes, to improve their homing potential towards an inflammatory site without affecting their behavior. The effect of HA-coatings on murine MSC was functionally determined both, in vitro and in vivo as a consequence of the transient CD44 overexpression induced by HA. Data obtained from the in vitro migration assay demonstrated a two-fold increase in the migratory potential of HA-treated MSC compared to untreated cells. In an LPS-induced inflamed ear murine model, HA-treated MSC demonstrated a significantly higher inflammatory targeting as observed at 72 hrs as compared to untreated cells. This increased accumulation for HA-treated MSC yielded a substantial reduction in inflammation as demonstrated by the decrease in the expression of pro-inflammatory markers and by the induction of a pro-regenerative environment.

Enhancing Vascularization through the Controlled Release of Platelet-Derived Growth Factor-BB.

Using delivery systems to control the in vivo release of growth factors (GFs) for tissue engineering applications is extremely desirable as the clinical use of GFs is limited by their fast in vivo turnover. Hence, the development of effective platforms that are able to finely control the release of GFs in vivo remains a challenge. Herein, we investigated the ability of multiscale microspheres, composed by a nanostructured silicon multistage vector (MSV) core and a poly(dl-lactide-co-glycolide) acid (PLGA) forming outer shell (PLGA-MSV), to release functional platelet-derived growth factor-BB (PDGF-BB) to induce in vivo localized neovascularization. The in vitro release of PDGF-BB was assessed by enzyme-linked immunosorbent assay (ELISA) over 2 weeks and showed a sustained, zero-order release kinetics. The ability to promote in vivo localized neovascularization was investigated in a subcutaneous injection model in BALB/c mice and followed by intravital microscopy up to 2 weeks. Fully functional newly formed vessels were found within the area where PLGA-MSVs were localized and covered 3.0 ± 0.9 and 19 ± 5.1% at 7 and 14 days, respectively, showing a 6-fold increase in 1 week. The distribution of CD31+ and α-SMA+ cells was detected by immunofluorescence on harvested tissues. CD31 was significantly more expressed (4-fold increase) compared to the untreated control. Finally, the level of up-regulation of angiogenesis-associated genes (Vegfa, Vwf, and Col3a1) was assessed by q-PCR, resulting in a significantly higher expression where PLGA-MSVs were localized (Vegfa: 2.32 ± 0.50 at 7 days and 4.37 ± 0.75 at 14 days; Vwf: 4.13 ± 0.82 and 7.74 ± 0.91; Col3a1: 5.43 ± 0.37 and 6.66 ± 0.89). Altogether, our data supported the conclusion that the localized delivery of PDGF-BB from PLGA-MSVs induced the localized de novo formation of fully functional vessels in vivo. With this study, we demonstrated that PLGA-MSV holds promise for accomplishing the controlled localized in vivo release of GFs for the design of innovative tissue engineering strategies.