Bone Marrow Mobilization and Local Stromal Cell-Derived Factor-1α Delivery Enhances Nascent Supraspinatus Muscle Fiber Growth.

Rotator cuff tear is a significant problem that leads to poor clinical outcomes due to muscle degeneration after injury. The objective of this study was to synergistically increase the number of proregenerative cells recruited to injure rotator cuff muscle through a novel dual treatment system, consisting of a bone marrow mobilizing agent (VPC01091), hypothesized to "push" prohealing cells into the blood, and localized delivery of stromal cell-derived factor-1α (SDF-1α), to "pull" the cells to the injury site. Immediately after rotator cuff tendon injury in rat, the mobilizing agent was delivered systemically, and SDF-1α-loaded heparin-based microparticles were injected into the supraspinatus muscle. Regenerative and degenerative changes to supraspinatus muscle and the presence of inflammatory/immune cells, mesenchymal stem cells (MSCs), and satellite cells were assessed via flow cytometry and histology for up to 21 days. After dual treatment, significantly more MSCs (31.9 ± 8.0% single cells) and T lymphocytes (6.7 ± 4.3 per 20 × field of view) were observed in supraspinatus muscle 7 days after injury and treatment compared to injury alone (14.4 ± 6.5% single cells, 1.2 ± 0.7 per 20 × field of view), in addition to an elevated M2:M1 macrophage ratio (3.0 ± 0.5), an indicator of a proregenerative environment. These proregenerative cellular changes were accompanied by increased nascent fiber formation (indicated by embryonic myosin heavy chain staining) at day 7 compared to SDF-1α treatment alone, suggesting that this method may be a promising strategy to influence the early cellular response in muscle and promote a proregenerative microenvironment to increase muscle healing after severe rotator cuff tear.

Analyzing immune response to engineered hydrogels by hierarchical clustering of inflammatory cell subsets.

Understanding the immune response to hydrogel implantation is critical for the design of immunomodulatory biomaterials. To study the progression of inflammation around poly(ethylene glycol) hydrogels presenting Arg-Gly-Asp (RGD) peptides and vascular endothelial growth factor, we used temporal analysis of high-dimensional flow cytometry data paired with intravital imaging, immunohistochemistry, and multiplexed proteomic profiling. RGD-presenting hydrogels created a reparative microenvironment promoting CD206+ cellular infiltration and revascularization in wounded dorsal skin tissue. Unbiased clustering algorithms (SPADE) revealed significant phenotypic transition shifts as a function of the cell-adhesion hydrogel properties. SPADE identified an intermediate macrophage subset functionally regulating in vivo cytokine secretion that was preferentially recruited for RGD-presenting hydrogels, whereas dendritic cell subsets were preferentially recruited to RDG-presenting hydrogels. Last, RGD-presenting hydrogels controlled macrophage functional cytokine secretion to direct polarization and vascularization. Our studies show that unbiased clustering of single-cell data provides unbiased insights into the underlying immune response to engineered materials.

Characterizing human mesenchymal stromal cells' immune-modulatory potency using targeted lipidomic profiling of sphingolipids.

Cell therapies are expected to increase over the next decade owing to increasing demand for clinical applications. Mesenchymal stromal cells (MSCs) have been explored to treat a number of diseases, with some successes in early clinical trials. Despite early successes, poor MSC characterization results in lessened therapeutic capacity once in vivo. Here, we characterized MSCs derived from bone marrow (BM), adipose tissue and umbilical cord tissue for sphingolipids (SLs), a class of bioactive lipids, using liquid chromatography/tandem mass spectrometry. We found that ceramide levels differed based on the donor's sex in BM-MSCs. We detected fatty acyl chain variants in MSCs from all three sources. Linear discriminant analysis revealed that MSCs separated based on tissue source. Principal component analysis showed that interferon-γ-primed and unstimulated MSCs separated according to their SL signature. Lastly, we detected higher ceramide levels in low indoleamine 2,3-dioxygenase MSCs, indicating that sphingomyelinase or ceramidase enzymatic activity may be involved in their immune potency.

Synthetic Matrix Scaffolds Engineer the In Vivo Tumor Immune Microenvironment for Immunotherapy Screening.

Immunotherapy has emerged as one of the most powerful anti-cancer therapies but is stymied by the limits of existing preclinical models with respect to disease latency and reproducibility. Additionally, the influence of differing immune microenvironments within tumors observed clinically and associated with immunotherapeutic resistance cannot be tuned to facilitate drug testing workflows without changing model system or laborious genetic approaches. To address this testing platform gap in the immune oncology drug development pipeline, the authors deploy engineered biomaterials as scaffolds to increase tumor formation rate, decrease disease latency, and diminish variability of immune infiltrates into tumors formed from murine mammary carcinoma cell lines implanted into syngeneic mice. By altering synthetic gel formulations that reshape infiltrating immune cells within the tumor, responsiveness of the same tumor model to varying classes of cancer immunotherapies, including in situ vaccination with a molecular adjuvant and immune checkpoint blockade, diverge. These results demonstrate the significant role the local immune microenvironment plays in immunotherapeutic response. These engineered tumor immune microenvironments therefore improve upon the limitations of current breast tumor models used for immune oncology drug screening to enable immunotherapeutic testing relevant to the variability in tumor immune microenvironments underlying immunotherapeutic resistance seen in human patients.

Single-cell RNA-seq of out-of-thaw mesenchymal stromal cells shows tissue-of-origin differences and inter-donor cell-cycle variations.

BACKGROUND: Human Mesenchymal stromal cells (hMSCs) from various tissue sources are widely investigated in clinical trials. These MSCs are often administered to patients immediately after thawing the cryopreserved product (out-of-thaw), yet little is known about the single-cell transcriptomic landscape and tissue-specific differences of out-of-thaw human MSCs. METHODS: 13 hMSC samples derived from 10 "healthy" donors were used to assess donor variability and tissue-of-origin differences in single-cell gene expression profiles. hMSCs derived and expanded from the bone marrow (BM) or cord tissue (CT) underwent controlled-rate freezing for 24 h. Cells were then transferred to the vapor phase of liquid nitrogen for cryopreservation. hMSCs cryopreserved for at least one week, were characterized immediately after thawing using a droplet-based single-cell RNA sequencing method. Data analysis was performed with SC3 and SEURAT pipelines followed by gene ontology analysis. RESULTS: scRNA-seq analysis of the hMSCs revealed two major clusters of donor profiles, which differ in immune-signaling, cell surface properties, abundance of cell-cycle related transcripts, and metabolic pathways of interest. Within-sample transcriptomic heterogeneity is low. We identified numerous differentially expressed genes (DEGs) that are associated with various cellular functions, such as cytokine signaling, cell proliferation, cell adhesion, cholesterol/steroid biosynthesis, and regulation of apoptosis. Gene-set enrichment analyses indicated different functional pathways in BM vs. CT hMSCs. In addition, MSC-batches showed significant variations in cell cycle status, suggesting different proliferative vs. immunomodulatory potential. Several potential transcript-markers for tissue source differences were identified for further investigation in future studies. In functional assays, both BM and CT MSCs suppressed macrophage TNFα secretion upon interferon stimulation. However, differences between donors, tissue-of-origin, and cell cycle are evident in both TNF suppression and cytokine secretion. CONCLUSIONS: This study shows that donor differences in hMSC transcriptome are minor relative to the intrinsic differences in tissue-of-origin. hMSCs with different transcriptomic profiles showed potential differences in functional characteristics. These findings contribute to our understanding of tissue origin-based differences in out-of-thaw therapeutic hMSC products and assist in the identification of cells with immune-regulatory or survival potential from a heterogeneous MSC population. Our results form the basis of future studies in correlating single-cell transcriptomic markers with immunomodulatory functions.

Immunotherapy via PD-L1-presenting biomaterials leads to long-term islet graft survival.

Antibody-mediated immune checkpoint blockade is a transformative immunotherapy for cancer. These same mechanisms can be repurposed for the control of destructive alloreactive immune responses in the transplantation setting. Here, we implement a synthetic biomaterial platform for the local delivery of a chimeric streptavidin/programmed cell death-1 (SA-PD-L1) protein to direct "reprogramming" of local immune responses to transplanted pancreatic islets. Controlled presentation of SA-PD-L1 on the surface of poly(ethylene glycol) microgels improves local retention of the immunomodulatory agent over 3 weeks in vivo. Furthermore, local induction of allograft acceptance is achieved in a murine model of diabetes only when receiving the SA-PD-L1-presenting biomaterial in combination with a brief rapamycin treatment. Immune characterization revealed an increase in T regulatory and anergic cells after SA-PD-L1-microgel delivery, which was distinct from naïve and biomaterial alone microenvironments. Engineering the local microenvironment via biomaterial delivery of checkpoint proteins has the potential to advance cell-based therapies, avoiding the need for systemic chronic immunosuppression.

Integrin-specific hydrogels modulate transplanted human bone marrow-derived mesenchymal stem cell survival, engraftment, and reparative activities.

Stem cell therapies are limited by poor cell survival and engraftment. A hurdle to the use of materials for cell delivery is the lack of understanding of material properties that govern transplanted stem cell functionality. Here, we show that synthetic hydrogels presenting integrin-specific peptides enhance the survival, persistence, and osteo-reparative functions of human bone marrow-derived mesenchymal stem cells (hMSCs) transplanted in murine bone defects. Integrin-specific hydrogels regulate hMSC adhesion, paracrine signaling, and osteoblastic differentiation in vitro. Hydrogels presenting GFOGER, a peptide targeting α2ß1 integrin, prolong hMSC survival and engraftment in a segmental bone defect and result in improved bone repair compared to other peptides. Integrin-specific hydrogels have diverse pleiotropic effects on hMSC reparative activities, modulating in vitro cytokine secretion and in vivo gene expression for effectors associated with inflammation, vascularization, and bone formation. These results demonstrate that integrin-specific hydrogels improve tissue healing by directing hMSC survival, engraftment, and reparative activities.

Lysostaphin and BMP-2 co-delivery reduces S. aureus infection and regenerates critical-sized segmental bone defects.

Staphylococcus aureus is the most common pathogen associated with bacterial infections in orthopedic procedures. Infections often lead to implant failure and subsequent removal, motivating the development of bifunctional materials that both promote repair and prevent failure due to infection. Lysostaphin is an anti-staphylococcal enzyme resulting in bacterial lysis and biofilm reduction. Lysostaphin use is limited by the lack of effective delivery methods to provide sustained, high doses of enzyme to infection sites. We engineered a BMP-2-loaded lysostaphin-delivering hydrogel that simultaneously prevents S. aureus infection and repairs nonhealing segmental bone defects in the murine radius. Lysostaphin-delivering hydrogels eradicated S. aureus infection and resulted in mechanically competent bone. Cytokine and immune cell profiling demonstrated that lysostaphin-delivering hydrogels restored the local inflammatory environment to that of a sterile injury. These results show that BMP-2-loaded lysostaphin-delivering hydrogel therapy effectively eliminates S. aureus infection while simultaneously regenerating functional bone resulting in defect healing.

Improving hard palate wound healing using immune modulatory autotherapies.

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.

Novel Lipid Signaling Mediators for Mesenchymal Stem Cell Mobilization during Bone Repair.

INTRODUCTION: Mesenchymal stem and progenitor cells (MSCs), which normally reside in the bone marrow, are critical to bone health and can be recruited to sites of traumatic bone injury, contributing to new bone formation. The ability to control the trafficking of MSCs provides therapeutic potential for improving traumatic bone healing and therapy for genetic bone diseases such as hypophosphatasia. METHODS: In this study, we explored the sphingosine-1-phosphate (S1P) signaling axis as a means to control the mobilization of MSCs into blood and possibly to recruit MSCs enhancing bone growth. RESULTS: Loss of S1P receptor 3 (S1PR3) leads to an increase in circulating CD45-/CD29+/CD90+/Sca1 putative mesenchymal progenitor cells, suggesting that blocking S1PR3 may stimulate MSCs to leave the bone marrow. Antagonism of S1PR3 with the small molecule VPC01091 stimulated acute migration of CD45-/CD29+/CD90+/Sca1+ MSCs into the blood as early as 1.5 hours after treatment. VPC01091 administration also increased ectopic bone formation induced by BMP-2 and significantly increased new bone formation in critically sized rat cranial defects, suggesting that mobilized MSCs may home to injuries to contribute to healing. We also explored the possibility of combining S1P manipulation of endogenous host cell occupancy with exogenous MSC transplantation for potential use in combination therapies. Importantly, reducing niche occupancy of host MSCs with VPC01091 does not impede engraftment of exogenous MSCs. CONCLUSIONS: Our studies suggest that MSC mobilization through S1PR3 antagonism is a promising strategy for endogenous tissue engineering and improving MSC delivery to treat bone diseases.

Enrichment of endogenous fractalkine and anti-inflammatory cells via aptamer-functionalized hydrogels.

Early recruitment of non-classical monocytes and their macrophage derivatives is associated with augmented tissue repair and improved integration of biomaterial constructs. A promising therapeutic approach to recruit these subpopulations is by elevating local concentrations of chemoattractants such as fractalkine (FKN, CX3CL1). However, delivering recombinant or purified proteins is not ideal due to their short half-lives, suboptimal efficacy, immunogenic potential, batch variabilities, and cost. Here we report an approach to enrich endogenous FKN, obviating the need for delivery of exogenous proteins. In this study, modified FKN-binding-aptamers are integrated with poly(ethylene glycol) diacrylate to form aptamer-functionalized hydrogels ("aptagels") that localize, dramatically enrich and passively release FKN in vitro for at least one week. Implantation in a mouse model of excisional skin injury demonstrates that aptagels enrich endogenous FKN and stimulate significant local increases in Ly6CloCX3CR1hi non-classical monocytes and CD206+ M2-like macrophages. The results demonstrate that orchestrators of inflammation can be manipulated without delivery of foreign proteins or cells and FKN-aptamer functionalized biomaterials may be a promising approach to recruit anti-inflammatory subpopulations to sites of injury. Aptagels are readily synthesized, highly customizable and could combine different aptamers to treat complex diseases in which regulation or enrichment of multiple proteins may be therapeutic.

Non-classical monocytes are biased progenitors of wound healing macrophages during soft tissue injury.

Successful tissue repair requires the activities of myeloid cells such as monocytes and macrophages that guide the progression of inflammation and healing outcome. Immunoregenerative materials leverage the function of endogenous immune cells to orchestrate complex mechanisms of repair; however, a deeper understanding of innate immune cell function in inflamed tissues and their subsequent interactions with implanted materials is necessary to guide the design of these materials. Blood monocytes exist in two primary subpopulations, characterized as classical inflammatory or non-classical. While classical monocytes extravasate into inflamed tissue and give rise to macrophages or dendritic cells, the recruitment kinetics and functional role of non-classical monocytes remains unclear. Here, we demonstrate that circulating non-classical monocytes are directly recruited to polymer films within skin injuries, where they home to a perivascular niche and generate alternatively activated, wound healing macrophages. Selective labeling of blood monocyte subsets indicates that non-classical monocytes are biased progenitors of alternatively activated macrophages. On-site delivery of the immunomodulatory small molecule FTY720 recruits S1PR3-expressing non-classical monocytes that support vascular remodeling after injury. These results elucidate a previously unknown role for blood-derived non-classical monocytes as contributors to alternatively activated macrophages, highlighting them as key regulators of inflammatory response and regenerative outcome.

Sphingosine-1-Phosphate Receptor-3 Supports Hematopoietic Stem and Progenitor Cell Residence Within the Bone Marrow Niche.

Hematopoietic stem and progenitor cells (HSPCs) egress from bone marrow (BM) during homeostasis and at increased rates during stress; however, the mechanisms regulating their trafficking remain incompletely understood. Here we describe a novel role for lipid receptor, sphingosine-1-phosphate receptor 3 (S1PR3), in HSPC residence within the BM niche. HSPCs expressed increased levels of S1PR3 compared to differentiated BM cells. Pharmacological antagonism or knockout (KO) of S1PR3 mobilized HSPCs into blood circulation, suggesting that S1PR3 influences niche localization. S1PR3 antagonism suppressed BM and plasma SDF-1, enabling HSPCs to migrate toward S1P-rich plasma. Mobilization synergized with AMD3100-mediated antagonism of CXCR4, which tethers HSPCs in the niche, and recovered homing deficits of AMD3100-treated grafts. S1PR3 antagonism combined with AMD3100 improved re-engraftment and survival in lethally irradiated recipients. Our studies indicate that S1PR3 and CXCR4 signaling cooperate to maintain HSPCs within the niche under homeostasis. These results highlight an important role for S1PR3 in HSPC niche occupancy and trafficking that can be harnessed for both rapid clinical stem cell mobilization and re-engraftment strategies, as well as the opportunity to design novel therapeutics for control of recruitment, homing, and localization through bioactive lipid signaling. Stem Cells 2017;35:1040-1052.

Monocytes and macrophages in tissue repair: Implications for immunoregenerative biomaterial design.

Monocytes and macrophages play a critical role in tissue development, homeostasis, and injury repair. These innate immune cells participate in guiding vascular remodeling, stimulation of local stem and progenitor cells, and structural repair of tissues such as muscle and bone. Therefore, there is a great interest in harnessing this powerful endogenous cell source for therapeutic regeneration through immunoregenerative biomaterial engineering. These materials seek to harness specific subpopulations of monocytes/macrophages to promote repair by influencing their recruitment, positioning, differentiation, and function within a damaged tissue. Monocyte and macrophage phenotypes span a continuum of inflammatory (M1) to anti-inflammatory or pro-regenerative cells (M2), and their heterogeneous functions are highly dependent on microenvironmental cues within the injury niche. Increasing evidence suggests that division of labor among subpopulations of monocytes and macrophages could allow for harnessing regenerative functions over inflammatory functions of myeloid cells; however, the complex balance between necessary functions of inflammatory versus regenerative myeloid cells remains to be fully elucidated. Historically, biomaterial-based therapies for promoting tissue regeneration were designed to minimize the host inflammatory response; although, recent appreciation for the roles that innate immune cells play in tissue repair and material integration has shifted this paradigm. A number of opportunities exist to exploit known signaling systems of specific populations of monocytes/macrophages to promote repair and to better understand the biological and pathological roles of myeloid cells. This review seeks to outline the characteristics of distinct populations of monocytes and macrophages, identify the role of these cells within diverse tissue injury niches, and offer design criteria for immunoregenerative biomaterials given the intrinsic inflammatory response to their implantation.

Bioactive lipid coating of bone allografts directs engraftment and fate determination of bone marrow-derived cells in rat GFP chimeras.

Bone grafting procedures are performed to treat wounds incurred during wartime trauma, accidents, and tumor resections. Endogenous mechanisms of repair are often insufficient to ensure integration between host and donor bone and subsequent restoration of function. We investigated the role that bone marrow-derived cells play in bone regeneration and sought to increase their contributions by functionalizing bone allografts with bioactive lipid coatings. Polymer-coated allografts were used to locally deliver the immunomodulatory small molecule FTY720 in tibial defects created in rat bone marrow chimeras containing genetically-labeled bone marrow for monitoring cell origin and fate. Donor bone marrow contributed significantly to both myeloid and osteogenic cells in remodeling tissue surrounding allografts. FTY720 coatings altered the phenotype of immune cells two weeks post-injury, which was associated with increased vascularization and bone formation surrounding allografts. Consequently, degradable polymer coating strategies that deliver small molecule growth factors such as FTY720 represent a novel therapeutic strategy for harnessing endogenous bone marrow-derived progenitors and enhancing healing in load-bearing bone defects.

Abluminal stimulation of sphingosine 1-phosphate receptors 1 and 3 promotes and stabilizes endothelial sprout formation.

Local delivery of lipid mediators has become a promising new approach for therapeutic angiogenesis and regenerative medicine. In this study, we investigated how gradient stimulation (either abluminal/distal or luminal/proximal) of engineered microvessels with sphingosine 1-phosphate (S1P) receptor-subtype-targeted molecules affects endothelial sprout growth using a microfluidic device. Our studies show that distal stimulation of microvessels with FTY720, an S1P1/3 selective agonist, promotes both arterial and venular sprout growth, whereas proximal stimulation does not. Using novel pharmacological antagonists of S1P receptor subtypes, we further show that S1P3 functionality is necessary for VEGF-induced sprouting, and confirmed these findings ex vivo using a murine aortic ring assay from S1P3-deficient mice. S1P3 agonist stimulation enhanced vascular stability in both cell types via upregulation of the interendothelial junction protein VE-cadherin. Lastly, S1P3 activation under flow promoted endothelial sprouting and branching while decreasing migratory cell fate in the microfluidic device. We used an in vivo murine dorsal skinfold window chamber model to confirm S1P3's role in neovascular branching. Together, these data suggest that a distal transendothelial gradient of S1P1/3-targeted drugs is an effective technique for both enhancing and stabilizing capillary morphogenesis in angiogenic applications.

Laminin- and basement membrane-polycaprolactone blend nanofibers as a scaffold for regenerative medicine.

Mimicking one or more components of the basement membrane (BM) holds great promise for overcoming insufficiencies in tissue engineering therapies. We have electrospun laminin nanofibers (NFs) isolated from the murine Engelbreth-Holm Swarm (EHS) tumor and evaluated them as a scaffold for embryonic stem cell culture. Seeded human embryonic stem cells were found to better maintain their undifferentiated, colony environment when cultured on laminin NFs compared to laminin mats, with 75% remaining undifferentiated on NFs. Mouse embryonic stem cells cultured on 10% laminin-polycaprolactone (PCL) NFs maintained their colony formation for twice as long without passage compared to those on PCL or gelatin substrates. In addition, we have established a protocol for electrospinning reconstituted basement membrane aligned (RBM)-PCL NFs within 10° of angular deviation. Neuron-like PC12 cells show significantly greater attachment (p < 0.001) and percentage of neurite-extending cells in vitro on 10% RBM-PCL NFs when compared to 1% and 0% RBM-PCL NFs (p < 0.015 and p < 0.001, respectively). Together, these results implicate laminin- and RBM-PCL scaffolds as a promising biomimetic substrate for regenerative medicine applications.

Multilayer cell-seeded polymer nanofiber constructs for soft-tissue reconstruction.

IMPORTANCE: Cell seeding throughout the thickness of a nanofiber construct allows for patient-specific implant alternatives with long-lasting effects, earlier integration, and reduced inflammation when compared with traditional implants. Cell seeding may improve implant integration with host tissue; however, the effect of cell seeding on thick nanofiber constructs has not been studied. OBJECTIVE: To use a novel cell-preseeded nanofiber tissue engineering technique to create a 3-dimensional biocompatible implant alternative to decellularized extracellular matrix. DESIGN: Animal study with mammalian cell culture to study tissue engineered scaffolds. SETTING: Academic research laboratory. PARTICIPANTS: Thirty-six Sprague-Dawley rats. INTERVENTIONS: The rats each received 4 implant types. The grafts included rat primary (enhanced green fluorescent protein-positive [eGFP+]) fibroblast-seeded polycaprolactone (PCL)/collagen nanofiber scaffold, PCL/collagen cell-free nanofiber scaffold, acellular human cadaveric dermis (AlloDerm), and acellular porcine dermis (ENDURAGen). Rats were monitored postoperatively and received enrofloxacin in the drinking water for 4 days prophylactically and buprenorphine (0.2-0.5 mg/kg administered subcutaneously twice a day postoperatively for pain for 48 hours). MAIN OUTCOMES AND MEASURES: The viability of NIH/3T3 fibroblasts cultured on PCL electrospun nanofibers was evaluated using fluorescence microscopy. Soft-tissue remodeling was examined histologically and with novel ex vivo volume determinations of implants using micro-computed tomography of cell-seeded implants relative to nanofibers without cells and commonly used dermal grafts of porcine and human origin (ENDURAGen and AlloDerm, respectively). The fate and distribution of eGFP+ seeded donor fibroblasts were assessed using immunohistochemistry. RESULTS: Fibroblasts migrated across nanofiber layers within 12 hours and remained viable on a single layer for up to 14 days. Scanning electron microscopy confirmed a nanoscale structure with a mean (SD) diameter of 158 (72) nm. Low extrusion rates demonstrated the excellent biocompatibility in vivo. Histological examination of the scaffolds demonstrated minimal inflammation. Cell seeding encouraged rapid vascularization of the nanofiber implants. Cells of donor origin (eGFP+) declined with the duration of implantation. Implant volume was not significantly affected for up to 8 weeks by the preseeding of cells (P > .05). CONCLUSIONS AND RELEVANCE: Polymer nanofiber-based scaffolds mimic natural extracellular matrix. Preseeding the nanofiber construct with cells improved vascularization without notable effects on volume. An effect of cell preseeding on scaffold vascularization was evident beyond the presence of preseeded cells. This 3-dimensional, multilayer method of cell seeding throughout a 1-mm-thick construct is simple and feasible for clinical application. Further development of this technique may affect the clinical practice of facial plastic and reconstructive surgeons.

The promotion of mandibular defect healing by the targeting of S1P receptors and the recruitment of alternatively activated macrophages.

Endogenous signals originating at the site of injury are involved in the paracrine recruitment, proliferation, and differentiation of circulating progenitor and diverse inflammatory cell types. Here, we investigate a strategy to exploit endogenous cell recruitment mechanisms to regenerate injured bone by local targeting and activation of sphingosine-1-phosphate (S1P) receptors. A mandibular defect model was selected for evaluating regeneration of bone following trauma or congenital disease. The particular challenges of mandibular reconstruction are inherent in the complex anatomy and function of the bone given that the area is highly vascularized and in close proximity to muscle. Nanofibers composed of poly(DL-lactide-co-glycolide) (PLAGA) and polycaprolactone (PCL) were used to delivery FTY720, a targeted agonist of S1P receptors 1 and 3. In vitro culture of bone progenitor cells on drug-loaded constructs significantly enhanced SDF1α mediated chemotaxis of bone marrow mononuclear cells. In vivo results show that local delivery of FTY720 from composite nanofibers enhanced blood vessel ingrowth and increased recruitment of M2 alternatively activated macrophages, leading to significant osseous tissue ingrowth into critical sized defects after 12 weeks of treatment. These results demonstrate that local activation of S1P receptors is a regenerative cue resulting in recruitment of wound healing or anti-inflammatory macrophages and bone healing. Use of such small molecule therapy can provide an alternative to biological factors for the clinical treatment of critical size craniofacial defects.

Regulation of osteoclast structure and function by FAK family kinases.

Osteoclasts are highly specialized cells that resorb bone and contribute to bone remodeling. Diseases such as osteoporosis and osteolytic bone metastasis occur when osteoclast-mediated bone resorption takes place in the absence of concurrent bone synthesis. Considerable effort has been placed on identifying molecules that regulate the bone resorption activity of osteoclasts. To this end, we investigated unique and overlapping functions of members of the FAK family (FAK and Pyk2) in osteoclast functions. With the use of a conditional knockout mouse model, in which FAK is selectively targeted for deletion in osteoclast precursors (FAK(Δmyeloid)), we found that loss of FAK resulted in reduced bone resorption by osteoclasts in vitro, coincident with impaired signaling through the CSF-1R. However, bone architecture appeared normal in FAK(Δmyeloid) mice, suggesting that Pyk2 might functionally compensate for reduced FAK levels in vivo. This was supported by data showing that podosome adhesion structures, which are essential for bone degradation, were significantly more impaired in osteoclasts when FAK and Pyk2 were reduced than when either molecule was depleted individually. We conclude that FAK contributes to cytokine signaling and bone resorption in osteoclasts and partially compensates for the absence of Pyk2 to maintain proper adhesion structures in these cells.