Evaluation of nanoscale versus hybrid micro/nano surface topographies for endosseous implants.

We examined the effect of a nanoscale titanium surface topography (D) versus two hybrid micro/nanoscale topographies (B and OS) on adherent mesenchymal stem cells (MSCs) and bone marrow derived macrophages (BMMs) function in cell culture and in vivo. In the in vitro study, compared to OS and B surfaces, D surface induced earlier and greater cell spreading, and earlier and profound mRNA expression of RUNX2, Osterix and BMP2 in MSCs. D surface induced earlier and higher expression of RUNX2 and BMP2 and lower expression of inflammatory genes in implant adherent cells in vivo. Measurement of osteogenesis at implant surfaces showed greater bone-to-implant contact at D versus OS surfaces after 21 days. We explored the cell population on the D and OS implant surfaces 24 h after placement using single-cell RNA sequencing and identified distinct cell clusters including macrophages, neutrophils and B cells. D surface induced lower expression and earlier reduction of inflammatory genes expression in BMMs in vitro. BMMs on D, B and OS surfaces demonstrated a marked increase of BMP2 expression after 1 and 3 days, and this increase was significantly higher on D surface at day 3. Our data implicates a dynamic process that may be influenced by nanotopography at multiple stages of osseointegration including initial immunomodulation, recruitment of MSCs and later osteoblastic differentiation leading to bone matrix production and mineralization. The results suggest that a nanoscale topography (D) favorably modulates adherent macrophage polarization toward anti-inflammatory and regenerative phenotypes and promotes the osteoinductive phenotype of adherent mesenchymal stem cells. STATEMENT OF SIGNIFICANCE: Our manuscript contains original data developed to define effects of a novel nanotopography on the process of osseointegration at the cell and tissue level.  Few studies have compared the effects of a nanoscale surface versus the more typical hybrid micro/nano-scale surfaces used today. We have utilized single-cell RNA sequencing for the first time to identify earliest cell populations on implant surfaces in vivo. We provide data indicating that the nanoscale surface acts upon both osteoprogenitor and immune cell (macrophages) to alter the process of bone formation in a surface-specific manner. This work represents new observations regarding osseointegration and immunomodulation.

Comparative Osteogenesis of Three Clinical Bone Graft Materials: An In Vivo Study.

PURPOSE: To investigate bone regeneration among three different bone graft materials in a rat calvarum model. MATERIALS AND METHODS: A total of 24 rats had two 5-mm defects placed per calvarial. Rats were divided into four groups: bovine xenograft (XG), demineralized bone matrix (DBM), mineralized bone graft (MBG), and collagen membrane control (CC). Within each group, samples were collected at two time points: 4 weeks (T4) and 8 weeks (T8). Bone regeneration was assessed by microcomputed tomography (micro-CT) imaging and was analyzed using MATLAB software. Additionally, the fixed samples were subsequently demineralized for immunohistochemistry and histomorphometry. Slides were mounted and stained with hematoxylin and eosin (H&E) stain as well as bone morphogenetic protein 2 (BMP-2) and runt-related transcription factor 2 (RUNX2) markers. The numbers of positive cells/area were calculated for each group and analyzed. RESULTS: At 4 weeks, DBM showed low mineral density (7.7%) compared to the control (25.2%), but increased dramatically at 8 weeks (DBM, T8 = 27.6%; CC, T8 = 27.2%). Xenograft material showed an increase in mineral desnity between T4 and T8 (XG, T4 = 25.0%; XG, T8 = 32.3%). MBG remained consistent over the 8-week trial period (MBG, T4 = 30.4%; MBG, T8 = 30.4%). BMP-2 expression was present in cells adherent to all graft materials. RUNX2 expression was also observed in cells adherent to all graft materials, indicating that during the 4- to 8-week healing period, all materials supported osteogenesis. CONCLUSIONS: Compared to other materials, the DBM had high osteoinductive properties during the 4- to 8-week time period based on increased mineral content. All materials were associated with immunohistologic evidence of osteogenesis in the rat calvarial defect model.

Micro RNA based MSC EV engineering: Targeting the BMP2 cascade for bone repair.

Mesenchymal stem cell derived extracellular vesicles (MSC EVs) possess excellent immunomodulatory and therapeutic properties. While beneficial, from a translational perspective, extracellular vesicles with consistent functionality and target specificity are required to achieve the goals of precision medicine and tissue engineering. Prior research has identified that the miRNA composition of mesenchymal stem cell derived extracellular vesicles contributes significantly towards extracellular vesicles functionality. In this study, we hypothesized that mesenchymal stem cell derived extracellular vesicle functionality can be rendered pathway-specific using a miRNA-based extracellular vesicles engineering approach. To test this hypothesis, we utilized bone repair as a model system and the BMP2 signaling cascade as the targeted pathway. We engineered mesenchymal stem cell extracellular vesicles to possess increased levels of miR-424, a potentiator of the BMP2 signaling cascade. We evaluated the physical and functional characteristics of these extracellular vesicles and their enhanced ability to trigger the osteogenic differentiation of naïve mesenchymal stem cell in vitro and facilitate bone repair in vivo. Results indicated that the engineered extracellular vesicles retained their extracellular vesicles characteristics and endocytic functionality and demonstrated enhanced osteoinductive function by activating SMAD1/5/8 phosphorylation and mesenchymal stem cell differentiation in vitro and enhanced bone repair in vivo. Furthermore, the inherent immunomodulatory properties of the mesenchymal stem cell derived extracellular vesicles remained unaltered. These results serve as a proof-of-concept for miRNA-based extracellular vesicles engineering approaches for regenerative medicine applications.

Extracellular Vesicles From TNFα Preconditioned MSCs: Effects on Immunomodulation and Bone Regeneration.

Mesenchymal stem cells show remarkable versatility and respond to extracellular and micro environmental cues by altering their phenotype and behavior. In this regard, the MSC's immunomodulatory properties in tissue repair are well documented. The paracrine effects of MSCs in immunomodulation are, in part, attributable to their secreted extracellular vesicles (EVs). When MSCs migrate to the wound bed, they are exposed to a myriad of inflammatory signals. To understand their response to an inflammatory environment from an EV perspective, we sought to evaluate the effects of the inflammatory cytokine TNFα on MSC EV mediated immunomodulation. Our results indicate that while the physical characteristics of the EVs remain unchanged, the TNFα preconditioned MSC EVs possess enhanced immunomodulatory properties. In vitro experiments using polarized (M1 and M2) primary mouse macrophages indicated that the preconditioned MSC EVs suppressed pro-inflammatory (M1) markers such as IL-1ß and iNOS and elevated reparatory (M2) markers such as Arg1 and CD206. When evaluated in vivo in a rat calvarial defect model, the TNFα preconditioned MSC EVs reduced inflammation at 1-, 3- and 7-days post wounding resulting in the subsequent enhanced bone formation at 4- and 8-weeks post wounding possibly by modulation of oncostatin M (OSM) expression. An analysis of EV miRNA composition revealed significant changes to anti-inflammatory miRNAs in the preconditioned MSC EVs hinting at a possible role for EV derived miRNA in the enhanced immunomodulatory activity. Overall, these results indicate that MSC exposure to inflammatory signals influence the MSC EV's immunomodulatory function in the context of tissue repair. The specific function of TNFα preconditioned MSC EV miRNAs in immunomodulatory control of bone regeneration merits further investigation.

3D Encapsulation and tethering of functionally engineered extracellular vesicles to hydrogels.

Mesenchymal stem cell (MSC) derived extracellular vesicles (EVs) in their naïve and engineered forms have emerged as potential alternatives to stem cell therapy. While they have a defined therapeutic potential, the spatial and temporal control of their activity in vivo remains a challenge. The objective of this study was to devise a methodology to encapsulate EVs in 3D hydrogels for prolonged delivery. To achieve this, we have leveraged the MSC EV interactions with ECM proteins and their derivative peptides. Using osteoinductive functionally engineered EVs (FEEs) derived from MSCs, we show that FEEs bind to mimetic peptides from collagen (DGEA, GFPGER) and fibronectin (RGD). In in vitro experiments, photocrosslinkable alginate hydrogels containing RGD were able to encapsulate, tether and retain the FEEs over a period of 7 days while maintaining the structural integrity and osteoinductive functionality of the EVs. When employed in a calvarial defect model in vivo, alginate-RGD hydrogels containing the FEEs enhanced bone regeneration by a factor of 4 compared to controls lacking FEEs and by a factor of 2 compared to controls lacking the tethering peptide. These results show that EVs can be tethered to biomaterials to promote bone repair and the importance of prolonged delivery in vivo. Results also provide a prelude to the possible use of this technology for controlled delivery of EVs for other regenerative medicine applications. STATEMENT OF SIGNIFICANCE: The beneficial effects of human MSC (HMSC) therapy are attributable to paracrine effects of the HMSC derived EVs. While EV engineering has the potential to impact several fields of regenerative medicine, targeted delivery of the engineered EVs with spatial and temporal control is necessary to prevent off-target effects and enhance tissue specificity. Here, we have leveraged the interactions of MSC EVs with ECM proteins to develop a tethering system that can be utilized to prolong EV delivery in vivo while maintaining the structural and functional integrity of the EVs. Our work has provided a tunable platform for EV delivery that we envision can be formulated as an injectable material or a bulk hydrogel suitable for regenerative medicine applications.

The importance of cellular and exosomal miRNAs in mesenchymal stem cell osteoblastic differentiation.

The differentiation of osteoblasts is under complex regulation that includes autocrine and paracrine signaling from MSCs. Exosomes are important components of the MSC secretome and their cargo contains numerous miRNAs. In this study, the importance of MSC miRNAs in modulation of osteoblastic differentiation was examined by global reduction of miRNA biosynthesis in Dicer knock down hMSCs. We additionally impaired hMSC responses to miRNAs by knockdown of Argonaute 2 expression. Knockdown of Dicer and Argonaute 2 both reduced osteoblastic differentiation of hMSCs. This was observed at the levels of hMSC culture mineralization and osteoblastic gene expression. The treatment of Dicer deficient hMSCs with wild type hMSC exosomes effectively recovered the impaired osteoblastic differentiation. Dicer knockdown reduced the quantity and diversity of miRNAs present in hMSC exosomes. miRSeq data and KEGG analysis implicated the miRNA-dependent effects on multiple osteoinductive pathways in Dicer deficient cells, including the Hippo signaling and TGF-beta signaling pathways. Treatment of hMSCs with mimics of miRNAs significantly downregulated in Dicer knockdown cells recovered functions of exosome-mediated signaling in hMSCs. These results indicate that hMSC exosomes exert miRNA-dependent control that contributes to osteoblastic differentiation.

Bone regeneration is mediated by macrophage extracellular vesicles.

Multiple local and systemic factors including inflammation influence bone regeneration. Several lines of evidence demonstrate that macrophages contribute to the immunological regulation of MSC and osteoblast function during bone regeneration. Recent studies demonstrate that macrophage polarization influences this regulatory process. In this manuscript, we investigated the paracrine functional role of naïve (M0), M1 and M2 polarized macrophage derived EVs in bone repair. Treatment of rat calvaria defects with no EVs, M0 EVs, M1 EVs, or M2 EVs revealed polarization-specific control of bone regeneration by macrophage EVs at 3 and 6 weeks. M0 and M2 EVs promoted repair/regeneration and M1 EVs inhibited bone repair. Pathway-specific studies conducted in cell culture showed that M1 EVs negatively regulated the BMP signaling pathway, specifically BMP2 and BMP9. In parallel, miRNA sequencing studies showed similar miRNA cargo in M0 and M2 EVs and different miRNA cargo in M1 EVs. Functional examination of M1 macrophage EV-enriched miR-155 demonstrated that miR-155 mimic treatment reduced MSC osteogenic differentiation as measured by reduced BMP2, BMP9 and RUNX2 expression when compared to controls. Conversely, treatment of MSCs with the M2 macrophage EV-enriched miR-378a mimic increased MSC osteoinductive gene expression when compared to controls. These functional studies implicate polarized macrophage EV miRNAs in the positive or negative regulation of bone regeneration that was observed in vivo. Overall, the results presented in this study indicate that macrophage polarization influences EV cargo and related EV function in the paracrine regulation of bone regeneration.

Functionally engineered extracellular vesicles improve bone regeneration.

Lineage specific differentiation of host mesenchymal stem cells (MSCs) is a necessary step for bone repair/regeneration. Clinically, growth factors such as bone morphogenetic protein 2 (BMP2) are used to enhance/hasten this process to heal critical sized defects. However, the clinical application of such growth factors is fraught with dosage challenges as well as immunological and ectopic complications. The identification of extracellular vesicles (EVs) as active components of the MSC secretome suggest alternative approaches to enhancing bone regeneration. Based on our earlier studies on the properties of EVs from lineage specified MSCs, this study sought to engineer EVs to enhance osteogenic differentiation. To generate MSC EVs with enhanced osteoinductive abilities, genetically modified human bone marrow derived MSCs (HMSCs) were generated by constitutively expressing BMP2. We hypothesized that these cells would generate functionally engineered EVs (FEEs) with enhanced osteoinductive properties. Our results show that these FEEs maintained the general physical and biochemical characteristics of naïve HMSC EVs in the form of size distribution, EV marker expression and endocytic properties but show increased bone regenerative potential compared to MSC EVs in a rat calvarial defect model in vivo. Mechanistic studies revealed that although BMP2 was constitutively expressed in the parental cells, the corresponding EVs (FEEs) do not contain BMP2 protein as an EV constituent. Further investigations revealed that the FEEs potentiate the BMP2 signaling cascade possibly due to an altered miRNA composition. Collectively, these studies indicate that EVs' functionality may be engineered by genetic modification of the parental MSCs to induce osteoinduction and bone regeneration. SIGNIFICANCE STATEMENT: With mounting evidence for the potential of MSC EVs in treatment of diseases and regeneration of tissues, it is imperative to evaluate if they can be modified for application specificity. The results presented here indicate the possibility for generating Functionally Engineered EVs (FEEs) from MSC sources. As a proof of concept approach, we have shown that EVs derived from genetically modified MSCs (BMP2 overexpression) can be effective as biomimetic substitutes for growth factors for enhanced tissue-specific regeneration (bone regeneration) in vivo. Mechanistic studies highlight the role of EV miRNAs in inducing pathway-specific changes. We believe that this study will be useful to researchers evaluating EVs for regenerative medicine applications.

Evaluating the Endocytosis and Lineage-Specification Properties of Mesenchymal Stem Cell Derived Extracellular Vesicles for Targeted Therapeutic Applications.

Mesenchymal stem cells (MSCs) are multipotent cells with regenerative and immunomodulatory properties. Several aspects of MSC function have been attributed to the paracrine effects of MSC derived extracellular vesicles (EVs). Although MSC EVs show great promise for regenerative medicine applications, insights into their uptake mechanisms by different target cells and the ability to control MSC EV properties for defined function in vivo have remained elusive knowledge gaps. The primary goal of this study is to elucidate how the basic properties of MSC derived EVs can be exploited for function-specific activity in regenerative medicine. Our first important observation is that, MSC EVs possess a common mechanism of endocytosis across multiple cell types. Second, altering the MSC state by inducing differentiation into multiple lineages did not affect the exosomal properties or endocytosis but triggered the expression of lineage-specific genes and proteins in vitro and in vivo respectively. Overall, the results presented in this study show a common mechanism of endocytosis for MSC EVs across different cell types and the feasibility to generate functionally enhanced EVs by modifications to parental MSCs.

Macrophage Control of Incipient Bone Formation in Diabetic Mice.

Both soft and hard tissue wound healing are impaired in diabetes. Diabetes negatively impacts fracture healing, bone regeneration and osseointegration of endosseous implants. The complex physiological changes associated with diabetes often manifest in immunological responses to wounding and repair where macrophages play a prominent role in determining outcomes. We hypothesized that macrophages in diabetes contribute toward impaired osseous wound healing. To test this hypothesis, we compared osseous wound healing in the mouse calvaria defect model using macrophages from C57BL/6J and db/db mice to direct osseous repair in both mouse strains. Initial analyses revealed that db/db mice macrophages showed an inflamed phenotype in its resting state. Incipient bone regeneration evaluated by µCT indicated that bone regeneration was relatively impaired in the db/db mouse calvaria and in the calvaria of C57BL/6J mice supplemented with db/db macrophages. Furthermore, osteogenic differentiation of mouse mesenchymal stem cells was negatively impacted by conditioned medium from db/db mice compared to C57BL/6J mice. Moreover, miR-Seq analysis revealed an altered miRNA composition in db/db macrophages with up regulated pro-inflammatory miRNAs and down regulated anti-inflammatory miRNAs. Overall, this study represents a direct step toward understanding macrophage-mediated regulation of osseous bone regeneration and its impairment in type 2 diabetes mellitus.

Engineering a Self-Assembling Leucine Zipper Hydrogel System with Function-Specific Motifs for Tissue Regeneration.

Protein-based self-assembling hydrogels can exhibit remarkably tunable properties as a scaffold for regenerative medicine applications. In this study, we sought to develop a leucine zipper (LZ) based self-assembling hydrogel with function-specific motifs for tissue-specific regeneration. As a proof-of-concept approach, we incorporated (a) calcium-binding domains ESQES and QESQSEQS derived from dentin matrix protein 1 (DMP1) and (b) an heparin-binding domain adjacent preceded by an MMP2 (matrix metalloprotease 2) cleavage site to facilitate loading of heparin binding growth factors, such as BMP-2, VEGF, and TGF-ß1, and their release in vivo by endogenous MMP2 proteolytic cleavage. These scaffolds were characterized and evaluated in vitro and in vivo. In vivo studies highlighted the potential of the engineered LZ hydrogel with respect to osteogenic differentiation of stem cells. The premineralized LZ scaffold loaded with HMSCs showed an enhanced osteoinductive property when compared with the control nonmineralized scaffold. The LZ backbone with heparin-binding domain containing an MMP2 cleavage site facilitated tethering of heparin-binding growth factors, such as VEGF, TGF-ß1 and BMP2 and demonstrated controlled release of these active growth factor both in vitro and in vivo and demonstrated growth factor specific activity in vivo (BMP-2 and TGF-ß1). Overall, we present a versatile protein based self-assembling system with tunable properties for tissue regeneration.

A Role for Exosomes in Craniofacial Tissue Engineering and Regeneration.

Tissue engineering and regenerative medicine utilize mesenchymal stem cells (MSCs) and their secretome in efforts to create or induce functional tissue replacement. Exosomes are specific extracellular vesicles (EVs) secreted by MSCs and other cells that carry informative cargo from the MSC to targeted cells that influence fundamental cellular processes including apoptosis, proliferation, migration, and lineage-specific differentiation. In this report, we review the current knowledge regarding MSC exosome biogenesis, cargo and function. This review summarizes the use of MSC exosomes to control or induce bone, cartilage, dentin, mucosa, and pulp tissue formation. The next-step engineering of exosomes provides additional avenues to enhance oral and craniofacial tissue engineering and regeneration.