Dynamic mass spectrometry probe for electrospray ionization mass spectrometry monitoring of bioreactors for therapeutic cell manufacturing.

Large-scale manufacturing of therapeutic cells requires bioreactor technologies with online feedback control enabled by monitoring of secreted biomolecular critical quality attributes (CQAs). Electrospray ionization mass spectrometry (ESI-MS) is a highly sensitive label-free method to detect and identify biomolecules, but requires extensive sample preparation before analysis, making online application of ESI-MS challenging. We present a microfabricated, monolithically integrated device capable of continuous sample collection, treatment, and direct infusion for ESI-MS detection of biomolecules in high-salt solutions. The dynamic mass spectrometry probe (DMSP) uses a microfluidic mass exchanger to rapidly condition samples for online MS analysis by removing interfering salts, while concurrently introducing MS signal enhancers to the sample for sensitive biomolecular detection. Exploiting this active conditioning capability increases MS signal intensity and signal-to-noise ratio. As a result, sensitivity for low-concentration biomolecules is significantly improved, and multiple proteins can be detected from chemically complex samples. Thus, the DMSP has significant potential to serve as an enabling portion of a novel analytical tool for discovery and monitoring of CQAs relevant to therapeutic cell manufacturing.

Do Surface Porosity and Pore Size Influence Mechanical Properties and Cellular Response to PEEK?

BACKGROUND: Despite its widespread use in orthopaedic implants such as soft tissue fasteners and spinal intervertebral implants, polyetheretherketone (PEEK) often suffers from poor osseointegration. Introducing porosity can overcome this limitation by encouraging bone ingrowth; however, the corresponding decrease in implant strength can potentially reduce the implant's ability to bear physiologic loads. We have previously shown, using a single pore size, that limiting porosity to the surface of PEEK implants preserves strength while supporting in vivo osseointegration. However, additional work is needed to investigate the effect of pore size on both the mechanical properties and cellular response to PEEK. QUESTIONS/PURPOSES: (1) Can surface porous PEEK (PEEK-SP) microstructure be reliably controlled? (2) What is the effect of pore size on the mechanical properties of PEEK-SP? (3) Do surface porosity and pore size influence the cellular response to PEEK? METHODS: PEEK-SP was created by extruding PEEK through NaCl crystals of three controlled ranges: 200 to 312, 312 to 425, and 425 to 508 µm. Micro-CT was used to characterize the microstructure of PEEK-SP. Tensile, fatigue, and interfacial shear tests were performed to compare the mechanical properties of PEEK-SP with injection-molded PEEK (PEEK-IM). The cellular response to PEEK-SP, assessed by proliferation, alkaline phosphatase activity, vascular endothelial growth factor production, and calcium content of osteoblast, mesenchymal stem cell, and preosteoblast (MC3T3-E1) cultures, was compared with that of machined smooth PEEK and Ti6Al4V. RESULTS: Micro-CT analysis showed that PEEK-SP layers possessed pores that were 284 ± 35 µm, 341 ± 49 µm, and 416 ± 54 µm for each pore size group. Porosity and pore layer depth ranged from 61% to 69% and 303 to 391 µm, respectively. Mechanical testing revealed tensile strengths > 67 MPa and interfacial shear strengths > 20 MPa for all three pore size groups. All PEEK-SP groups exhibited > 50% decrease in ductility compared with PEEK-IM and demonstrated fatigue strength > 38 MPa at one million cycles. All PEEK-SP groups also supported greater proliferation and cell-mediated mineralization compared with smooth PEEK and Ti6Al4V. CONCLUSIONS: The PEEK-SP formulations evaluated in this study maintained favorable mechanical properties that merit further investigation into their use in load-bearing orthopaedic applications and supported greater in vitro osteogenic differentiation compared with smooth PEEK and Ti6Al4V. These results are independent of pore sizes ranging 200 µm to 508 µm. CLINICAL RELEVANCE: PEEK-SP may provide enhanced osseointegration compared with current implants while maintaining the structural integrity to be considered for several load-bearing orthopaedic applications such as spinal fusion or soft tissue repair.

Rescue of a primary myelofibrosis model by retinoid-antagonist therapy.

Molecular targeting of the two receptor interaction domains of the epigenetic repressor silencing mediator of retinoid and thyroid hormone receptors (SMRT(mRID)) produced a transplantable skeletal syndrome that reduced radial bone growth, increased numbers of bone-resorbing periosteal osteoclasts, and increased bone fracture risk. Furthermore, SMRT(mRID) mice develop spontaneous primary myelofibrosis, a chronic, usually idiopathic disorder characterized by progressive bone marrow fibrosis. Frequently linked to polycythemia vera and chronic myeloid leukemia, myelofibrosis displays high patient morbidity and mortality, and current treatment is mostly palliative. To decipher the etiology of this disease, we identified the thrombopoietin (Tpo) gene as a target of the SMRT-retinoic acid receptor signaling pathway in bone marrow stromal cells. Chronic induction of Tpo in SMRT(mRID) mice results in up-regulation of TGF-ß and PDGF in megakaryocytes, uncontrolled proliferation of bone marrow reticular cells, and fibrosis of the marrow compartment. Of therapeutic relevance, we show that this syndrome can be rescued by retinoid antagonists, demonstrating that the physical interface between SMRT and retinoic acid receptor can be a potential therapeutic target to block primary myelofibrosis disease progression.

Hydrogel-based Delivery of rhBMP-2 Improves Healing of Large Bone Defects Compared With Autograft.

BACKGROUND: Autologous bone grafting remains the gold standard in the treatment of large bone defects but is limited by tissue availability and donor site morbidity. Recombinant human bone morphogenetic protein-2 (rhBMP-2), delivered with a collagen sponge, is clinically used to treat large bone defects and complications such as delayed healing or nonunion. For the same dose of rhBMP-2, we have shown that a hybrid nanofiber mesh-alginate (NMA-rhBMP-2) delivery system provides longer-term release and increases functional bone regeneration in critically sized rat femoral bone defects compared with a collagen sponge. However, no comparisons of healing efficiencies have been made thus far between this hybrid delivery system and the gold standard of using autograft. QUESTIONS/PURPOSES: We compared the efficacy of the NMA-rhBMP-2 hybrid delivery system to morselized autograft and hypothesized that the functional regeneration of large bone defects observed with sustained BMP delivery would be at least comparable to autograft treatment as measured by total bone volume and ex vivo mechanical properties. METHODS: Bilateral critically sized femoral bone defects in rats were treated with either live autograft or with the NMA-rhBMP-2 hybrid delivery system such that each animal received one treatment per leg. Healing was monitored by radiography and histology at 2, 4, 8, and 12 weeks. Defects were evaluated for bone formation by longitudinal micro-CT scans over 12 weeks (n = 14 per group). The bone volume, bone density, and the total new bone formed beyond 2 weeks within the defect were calculated from micro-CT reconstructions and values compared for the 2-, 4-, 8-, and 12-week scans within and across the two treatment groups. Two animals were used for bone labeling with subcutaneously injected dyes at 4, 8, and 12 weeks followed by histology at 12 weeks to identify incremental new bone formation. Functional recovery was measured by ex vivo biomechanical testing (n = 9 per group). Maximum torque and torsional stiffness calculated from torsion testing of the femurs at 12 weeks were compared between the two groups. RESULTS: The NMA-rhBMP-2 hybrid delivery system resulted in greater bone formation and improved biomechanical properties compared with autograft at 12 weeks. Comparing new bone volume within each group, the NMA-rhBMP-2-treated group had higher volume (p < 0.001) at 12 weeks (72.59 ± 18.34 mm(3)) compared with 8 weeks (54.90 ± 16.14) and 4 weeks (14.22 ± 9.59). The new bone volume was also higher at 8 weeks compared with 4 weeks (p < 0.001). The autograft group showed higher (p <0.05) new bone volume at 8 weeks (11.19 ± 8.59 mm(3)) and 12 weeks (14.64 ± 10.36) compared with 4 weeks (5.15 ± 4.90). Between groups, the NMA-rhBMP-2-treated group had higher (p < 0.001) new bone volume than the autograft group at both 8 and 12 weeks. Local mineralized matrix density in the NMA-rhBMP-2-treated group was lower than that of the autograft group at all time points (p < 0.001). Presence of nuclei within the lacunae of the autograft and early appositional bone formation seen in representative histology sections suggested that the bone grafts remained viable and were functionally engrafted within the defect. The bone label distribution from representative sections also revealed more diffuse mineralization in the defect in the NMA-rhBMP-2-treated group, whereas more localized distribution of new mineral was seen at the edges of the graft pieces in the autograft group. The NMA-rhBMP-2-treated group also revealed higher torsional stiffness (0.042 ± 0.019 versus 0.020 ± 0.022 N-m/°; p = 0.037) and higher maximum torque (0.270 ± 0.108 versus 0.125 ± 0.137 N-m; p = 0.024) compared with autograft. CONCLUSIONS: The NMA-rhBMP-2 hybrid delivery system improved bone formation and restoration of biomechanical function of rat segmental bone defects compared with autograft treatment. CLINICAL RELEVANCE: Delivery systems that allow prolonged availability of BMP may provide an effective clinical alternative to autograft treatment for repair of segmental bone defects. Future studies in a large animal model comparing mixed cortical-trabecular autograft and the NMA-rhBMP-2 hybrid delivery system are the next step toward clinical translation of this approach.

Mesenchymal Stromal Cell (MSC) Functional Analysis-Macrophage Activation and Polarization Assays.

Stem cell-based therapies have evolved to become a key component of regenerative medicine approaches to human pathologies. Exogenous stem cell transplantation takes advantage of the potential of stem cells to self-renew, differentiate, home to sites of injury, and sufficiently evade the immune system to remain viable for the release of anti-inflammatory cytokines, chemokines, and growth factors. Common to many pathologies is the exacerbation of inflammation at the injury site by proinflammatory macrophages. An increasing body of evidence has demonstrated that mesenchymal stromal cells (MSCs) can influence the immunophenotype and function of myeloid lineage cells to promote therapeutic effects. Understanding the degree to which MSCs can modulate the phenotype of macrophages within an inflammatory environment is of interest when considering strategies for targeted cell therapies. There is a critical need for potency assays to elucidate these intercellular interactions in vitro and provide insight into potential mechanisms of action attributable to the immunomodulatory and polarizing capacities of MSCs, as well as other cells with immunomodulatory potential. However, the complexity of the responses, in terms of cell phenotypes and characteristics, timing of these interactions, and the degree to which cell contact is involved, have made the study of these interactions challenging. To provide a research tool to study the direct interactions between MSCs and macrophages, we developed a potency assay that directly co-cultures MSCs with naïve macrophages under proinflammatory conditions. Using this assay, we demonstrated changes in the macrophage secretome and phenotype, which can be used to evaluate the abilities of the cell samples to influence the cell microenvironment. These results suggest the immunomodulatory effects of MSCs on macrophages while revealing key cytokines and phenotypic changes that may inform their efficacy as potential cellular therapies. Key features • The protocol uses monocytes differentiated into naïve macrophages, which are loosely adherent, have a relatively homogeneous genetic background, and resemble peripheral blood mononuclear cells-derived macrophages. • The protocol requires a plate reader and a flow cytometer with the ability to detect six fluorophores. • The protocol provides a quantitative measurement of co-culture conditions by the addition of a fixed number of freshly thawed or culture-rescued MSCs to macrophages. • This protocol uses assessment of the secretome and cell harvest to independently verify the nature of the interactions between macrophages and MSCs.

Human stem cell delivery for treatment of large segmental bone defects.

Local or systemic stem cell delivery has the potential to promote repair of a variety of damaged or degenerated tissues. Although various stem cell sources have been investigated for bone repair, few comparative reports exist, and cellular distribution and viability postimplantation remain key issues. In this study, we quantified the ability of tissue-engineered constructs containing either human fetal or adult stem cells to enhance functional repair of nude rat critically sized femoral defects. After 12 weeks, defects treated with cell-seeded polymer scaffolds had significantly higher bone ingrowth and torsional strength compared to those receiving acellular scaffolds, although there were no significant differences between the cell sources. Next, stem cells were labeled with fluorescent quantum dots (QDs) in an attempt to noninvasively track their distribution after delivery on scaffolds. Clear fluorescence was observed at implantation sites throughout the study; however, beginning 7-10 days after surgery, signals were also observed at contralateral sites treated with acellular QD-free scaffolds. Although immunostaining for human nuclei revealed retention of some cells at the implantation site, no human cells were detected in the control limb defects. Additional histological analysis of implantation and control defect tissues revealed macrophages containing endocytosed QDs. Furthermore, QD-labeling appeared to diminish transplanted cell function resulting in reduced healing responses. In summary, augmentation of polymeric scaffolds with stem cells derived from fetal and adult tissues significantly enhanced healing of large segmental bone defects; however, QD labeling of stem cells eliminated the observed therapeutic effect and failed to conclusively track stem cell location long-term in vivo.

Mesenchymal stromal cells for bone trauma, defects, and disease: Considerations for manufacturing, clinical translation, and effective treatments.

Bone is a complex tissue capable of natural repair to injury, however, the healing process is often impaired by the untoward effects of trauma, defects, and disease. Thus, therapeutic modalities, including the use of cells involved in the body's natural healing processes, are investigated to promote or complement natural bone repair. Herein, several modalities and innovative approaches for using mesenchymal stromal cells (MSCs) to treat bone trauma, defects, and diseases are discussed. Given the evidence that supports the promising potential of MSCs, we highlight important considerations for advancing the clinical use of MSCs including the standardization of procedures from the harvest to delivery to patients and realized solutions to manufacturing. A better understanding of the current approaches implemented to address the challenges of using therapeutic MSCs will help improve study designs and, ultimately, achieve effective outcomes for restoring bone health.

Intra-articular delivery of micronized dehydrated human amnion/chorion membrane reduces degenerative changes after onset of post-traumatic osteoarthritis.

Background: Micronized dehydrated human amnion/chorion membrane (mdHACM) has reduced short term post-traumatic osteoarthritis (PTOA) progression in rats when delivered 24 h after medial meniscal transection (MMT) and is being investigated for clinical use as a disease modifying therapy. Much remains to be assessed, including its potential for longer-term therapeutic benefit and treatment effects after onset of joint degeneration. Objectives: Characterize longer-term effects of acute treatment with mdHACM and determine whether treatment administered to joints with established PTOA could slow or reverse degeneration. Hypotheses: Acute treatment effects will be sustained for 6 weeks, and delivery of mdHACM after onset of joint degeneration will attenuate structural osteoarthritic changes. Methods: Rats underwent MMT or sham surgery (left leg). mdHACM was delivered intra-articularly 24 h or 3 weeks post-surgery (n = 5-7 per group). Six weeks post-surgery, animals were euthanized and left tibiae scanned using equilibrium partitioning of an ionic contrast agent microcomputed tomography (EPIC-µCT) to structurally quantify joint degeneration. Histology was performed to examine tibial plateau cartilage. Results: Quantitative 3D µCT showed that cartilage structural metrics (thickness, X-ray attenuation, surface roughness, exposed bone area) for delayed mdHACM treatment limbs were significantly improved over saline treatment and not significantly different from shams. Subchondral bone mineral density and thickness for the delayed treatment group were significantly improved over acute treated, and subchondral bone thickness was not significantly different from sham. Marginal osteophyte degenerative changes were decreased with delayed mdHACM treatment compared to saline. Acute treatment (24 h post-surgery) did not reduce longer-term joint tissue degeneration compared to saline. Histology supported µCT findings and further revealed that while delayed treatment reduced cartilage damage, chondrocytes displayed qualitatively different morphologies and density compared to sham. Conclusion: This study provides insight into effects of intra-articular delivery timing relative to PTOA progression and the duration of therapeutic benefit of mdHACM. Results suggest that mdHACM injection into already osteoarthritic joints can improve joint health, but a single, acute mdHACM injection post-injury does not prevent long term osteoarthritis associated with meniscal instability. Further work is needed to fully characterize the durability of therapeutic benefit in stable osteoarthritic joints and the effects of repeated injections.

Streamlined Methods for Processing and Cryopreservation of Cell Therapy Products Using Automated Systems.

Streamlined procedures for processing and cryopreservation of cell therapies using good laboratory practices are integral to biomanufacturing process development and clinical applications. The protocol herein begins with the preparation of human cell types cultured as adherent (i.e., mesenchymal stromal cells, MSCs) or suspension cells (i.e., peripheral blood mononuclear cells, PBMCs) to comprehensively demonstrate procedures that are applicable to commonly used primary cell cultures. Cell processing steps consist of preparing high yields of cells for cryopreservation using instruments routinely used in cell manufacturing, including the Finia® Fill and Finish System and a controlled-rate freezer. The final steps comprise the storage of cells at subzero temperatures in liquid nitrogen vapor phase followed by the analysis of cell phenotypes before and after processing and cryopreservation, along with cell quality metrics for validation. Additionally, the protocol includes important considerations for the implementation of quality control measures for equipment operation and cell handling, as well as Good Laboratory Practices for cell manufacturing, which are essential for the translational use of cell therapies. Key features • The protocol applies to small- or large-scale manufacturing of cell therapy products. • It includes streamlined procedures for processing and cryopreservation of cells cultured as adherent cells (MSCs) and suspension cells (PBMCs). • Provides temperature control and rapid partitioning of sample in cryopreservation solution to maintain high viability of a range of cell types throughout the procedures. • This protocol employs the Finia® Fill and Finish System and a controlled-rate freezer. Graphical overview.

Patient-Specific iPSC-Derived Models Link Aberrant Endoplasmic Reticulum Stress Sensing and Response to Juvenile Osteochondritis Dissecans Etiology.

Juvenile osteochondritis dissecans (JOCD) is a pediatric disease, which begins with an osteonecrotic lesion in the secondary ossification center which, over time, results in the separation of the necrotic fragment from the parent bone. JOCD predisposes to early-onset osteoarthritis. However, the knowledge gap in JOCD pathomechanisms severely limits current therapeutic strategies. To elucidate its etiology, we conducted a study with induced pluripotent stem cells (iPSCs) from JOCD and control patients. iPSCs from skin biopsies were differentiated to iMSCs (iPSC-derived mesenchymal stromal cells) and subjected to chondrogenic and endochondral ossification, and endoplasmic reticulum (ER)-stress induction assays. Our study, using 3 JOCD donors, showed that JOCD cells have lower chondrogenic capability and their endochondral ossification process differs from control cells; yet, JOCD- and control-cells accomplish osteogenesis of similar quality. Our findings show that endoplasmic reticulum stress sensing and response mechanisms in JOCD cells, which partially regulate chondrocyte and osteoblast differentiation, are related to these differences. We suggest that JOCD cells are more sensitive to ER stress than control cells, and in pathological microenvironments, such as microtrauma and micro-ischemia, JOCD pathogenesis pathways may be initiated. This study is the first, to the best of our knowledge, to realize the important role that resident cells and their differentiating counterparts play in JOCD and to put forth a novel etiological hypothesis that seeks to consolidate and explain previously postulated hypotheses. Furthermore, our results establish well-characterized JOCD-specific iPSC-derived in vitro models and identified potential targets which could be used to improve diagnostic tools and therapeutic strategies in JOCD.

Synthetic scaffold coating with adeno-associated virus encoding BMP2 to promote endogenous bone repair.

Biomaterial scaffolds functionalized to stimulate endogenous repair mechanisms via the incorporation of osteogenic cues offer a potential alternative to bone grafting for the treatment of large bone defects. We first quantified the ability of a self-complementary adeno-associated viral vector encoding bone morphogenetic protein 2 (scAAV2.5-BMP2) to enhance human stem cell osteogenic differentiation in vitro. In two-dimensional culture, scAAV2.5-BMP2-transduced human mesenchymal stem cells (hMSCs) displayed significant increases in BMP2 production and alkaline phosphatase activity compared with controls. hMSCs and human amniotic-fluid-derived stem cells (hAFS cells) seeded on scAAV2.5-BMP2-coated three-dimensional porous polymer Poly(ε-caprolactone) (PCL) scaffolds also displayed significant increases in BMP2 production compared with controls during 12 weeks of culture, although only hMSC-seeded scaffolds displayed significantly increased mineral formation. PCL scaffolds coated with scAAV2.5-BMP2 were implanted into critically sized immunocompromised rat femoral defects, both with or without pre-seeding of hMSCs, representing ex vivo and in vivo gene therapy treatments, respectively. After 12 weeks, defects treated with acellular scAAV2.5-BMP2-coated scaffolds displayed increased bony bridging and had significantly higher bone ingrowth and mechanical properties compared with controls, whereas defects treated with scAAV2.5-BMP2 scaffolds pre-seeded with hMSCs failed to display significant differences relative to controls. When pooled, defect treatment with scAAV2.5-BMP2-coated scaffolds, both with or without inclusion of pre-seeded hMSCs, led to significant increases in defect mineral formation at all time points and increased mechanical properties compared with controls. This study thus presents a novel acellular bone-graft-free endogenous repair therapy for orthotopic tissue-engineered bone regeneration.

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.

Molecular Crosstalk Between Macrophages and Mesenchymal Stromal Cells.

Mesenchymal stromal cells (MSCs) have been widely investigated for regenerative medicine applications, from treating various inflammatory diseases as a cell therapy to generating engineered tissue constructs. Numerous studies have evaluated the potential effects of MSCs following therapeutic administration. By responding to their surrounding microenvironment, MSCs may mediate immunomodulatory effects through various mechanisms that directly (i.e., contact-dependent) or indirectly (i.e., paracrine activity) alter the physiology of endogenous cells in various disease pathologies. More specifically, a pivotal crosstalk between MSCs and tissue-resident macrophages and monocytes (TMφ) has been elucidated using in vitro and in vivo preclinical studies. An improved understanding of this crosstalk could help elucidate potential mechanisms of action (MOAs) of therapeutically administered MSCs. TMφ, by nature of their remarkable functional plasticity and prevalence within the body, are uniquely positioned as critical modulators of the immune system - not only in maintaining homeostasis but also during pathogenesis. This has prompted further exploration into the cellular and molecular alterations to TMφ mediated by MSCs. In vitro assays and in vivo preclinical trials have identified key interactions mediated by MSCs that polarize the responses of TMφ from a pro-inflammatory (i.e., classical activation) to a more anti-inflammatory/reparative (i.e., alternative activation) phenotype and function. In this review, we describe physiological and pathological TMφ functions in response to various stimuli and discuss the evidence that suggest specific mechanisms through which MSCs may modulate TMφ phenotypes and functions, including paracrine interactions (e.g., secretome and extracellular vesicles), nanotube-mediated intercellular exchange, bioenergetics, and engulfment by macrophages. Continued efforts to elucidate this pivotal crosstalk may offer an improved understanding of the immunomodulatory capacity of MSCs and inform the development and testing of potential MOAs to support the therapeutic use of MSCs and MSC-derived products in various diseases.

Rapid changes in shear stress induce dissociation of a G alpha(q/11)-platelet endothelial cell adhesion molecule-1 complex.

It has been recently shown that endothelial platelet endothelial cell adhesion molecule-1 (PECAM-1) expression is pro-atherogenic. PECAM-1 is involved in sensing rapid changes in fluid shear stress but the mechanisms for activating signalling complexes at the endothelial cell junction have yet to be elucidated. Additional studies suggest the activation of membrane-bound G proteins G alpha(q/11) also mediate flow-induced responses. Here, we investigated whether PECAM-1 and G alpha(q/11) could act in unison to rapidly respond to fluid shear stress. With immunohistochemistry, we observed a co-localization of G alpha(q/11) and PECAM-1 at the cell-cell junction in the atheroprotected section of mouse aortae. In contrast, G alpha(q/11) was absent from junctions in atheroprone areas as well as in all arterial sections of PECAM-1 knockout mice. In primary human endothelial cells, temporal gradients in shear stress led to a rapid dissociation of the G alpha(q/11)-PECAM-1 complex within 30 s and a partial relocalization of the G alpha(q/11) staining to perinuclear areas within 150 min, whereas transitioning fluid flow devoid of temporal gradients did not disrupt the complex. Inhibition of G protein activation eliminated temporal gradient flow-induced G alpha(q/11)-PECAM-1 dissociation. These results allow us to conclude that G alpha(q/11)-PECAM-1 forms a mechanosensitive complex and its localization suggests the G alpha(q/11)-PECAM-1 complex is a critical mediator of vascular diseases.

The Effects of Age and Dose on Gene Expression and Segmental Bone Defect Repair After BMP-2 Delivery.

Age is a well-known influential factor in bone healing, with younger patients generally healing bone fractures more rapidly and suffering fewer complications compared with older patients. Yet the impact age has on the response to current bone healing treatments, such as delivery of bone morphogenetic protein 2 (BMP-2), remains poorly characterized. It remains unclear how or if therapeutic dosing of BMP-2 should be modified to account for age-related differences in order to minimize potential adverse effects and consequently improve patient bone-healing outcomes. For this study, we sought to address this issue by using a preclinical critically sized segmental bone defect model in rats to investigate age-related differences in bone repair after delivery of BMP-2 in a collagen sponge, the current clinical standard. Femoral defects were created in young (7-week-old) and adult (8-month-old) rats, and healing was assessed using gene expression analyses, longitudinal radiography, ex vivo micro-computed tomography (µCT), as well as torsional testing. We found that young rats demonstrated elevated expression of genes related to osteogenesis, chondrogenesis, and matrix remodeling at the early 1-week time point compared with adult rats. These early gene expression differences may have impacted long-term healing as the regenerated bones of young rats exhibited higher bone mineral densities compared with those of adult rats after 12 weeks. Furthermore, the young rats demonstrated significantly more bone formation and increased mechanical strength when BMP-2 dose was increased from 1 µg to 10 µg, a finding not observed in adult rats. Overall, these results indicate there are age-related differences in BMP-2-mediated bone regeneration, including relative dose sensitivity, suggesting that age is an important consideration when implementing a BMP-2 treatment strategy.

Effects of BMP-2 dose and delivery of microvascular fragments on healing of bone defects with concomitant volumetric muscle loss.

Traumatic composite bone-muscle injuries, such as open fractures, often require multiple surgical interventions and still typically result in long-term disability. Clinically, a critical indicator of composite injury severity is vascular integrity; vascular damage alone is sufficient to assign an open fracture to the most severe category. Challenging bone injuries are often treated with bone morphogenetic protein 2 (BMP-2), an osteoinductive growth factor, delivered on collagen sponge. Previous studies in a composite defect model found that a minimally bridging dose in the segmental defect model was unable to overcome concomitant muscle damage, but the effect of BMP dose on composite injuries has not yet been studied. Here, we test the hypotheses that BMP-2-mediated functional regeneration of composite extremity injuries is dose dependent and can be further enhanced via co-delivery of adipose-derived microvascular fragments (MVF), which have been previously shown to increase tissue vascular volume. Although MVF did not improve healing outcomes, we observed a significant BMP-2 dose-dependent increase in regenerated bone volume and biomechanical properties. This is the first known report of an increased BMP-2 dose improving bone healing with concomitant muscle damage. While high dose BMP-2 delivery can induce heterotopic ossification (HO) and increased inflammation, the maximum 10 µg dose used in this study did not result in HO and was associated with a lower circulating inflammatory cytokine profile than the low dose (2.5 µg) group. These data support the potential benefits of an increased, though still moderate, BMP-2 dose for treatment of bone defects with concomitant muscle damage. Published 2019. This article is a U.S. Government work and is in the public domain in the USA. J Orthop Res.

Aggregate mesenchymal stem cell delivery ameliorates the regenerative niche for muscle repair.

Duchenne muscular dystrophy is a severe muscle wasting disease due to the absence of the dystrophin protein from the muscle cell membrane, which renders the muscle susceptible to continuous damage. In Duchenne muscular dystrophy patients, muscle weakness, together with cycles of degeneration/regeneration and replacement with noncontractile tissue, limit mobility and lifespan. Because the loss of dystrophin results in loss of polarity and a reduction in the number of self-renewing satellite cells, it is postulated that these patients could achieve an improved quality of life if delivered cells could restore satellite cell function. In this study, we used both an established myotoxic injury model in wild-type (WT) mice and mdx mice alone (spontaneous muscle damage). Single (SC) and aggregated (AGG) mesenchymal stem cells (MSCs) were injected into the gastrocnemius muscles 4 hr after injury (WT mice). The recovery of peak isometric torque was longitudinally assessed over 5 weeks, with earlier takedowns for histological assessment of healing (fibre cross-section area and central nucleation) and MSC retention. AGG-treated WT mice had significantly greater torque recovery at Day 14 than SC or saline-treated mice and a greater CSA at Day 10, compared with SC/saline. AGG-treated mdx mice had a greater peak isometric torque compared with SC/saline. In vitro immunomodulatory factor secretion of AGG-MSCs was higher than SC-MSCs for all tested growth factors with the largest difference observed in hepatocyte growth factor. Future studies are necessary to pair immunomodulatory factor secretion with functional attributes, to better predict the potential therapeutic value of MSC treatment modalities.

Human platelet lysate supplementation of mesenchymal stromal cell delivery: issues of xenogenicity and species variability.

Immunogenicity of fetal bovine serum (FBS) poses a problem for its use in the propagation of autologous mesenchymal stromal cells (MSCs) for cell therapy. Human platelet lysate (hPL), an enriched growth factor solution containing mitogenic and angiogenic cues, has potential utility in replacing FBS for human MSC (hMSC) delivery strategies. Despite its potentiation of hMSC number in vitro, little is known concerning its capacity to supplement implanted hMSC-seeded constructs and promote tissue regeneration in vivo. In this study, we tested the effects of incorporating hPL in cell-seeded constructs implanted subcutaneously into immunocompromised rats, investigated in vitro interactions between hPL and rat MSCs (rMSCs) and determined interspecies variability in the PL product [hPL vs rat PL (rPL)] and its effect on cultured MSCs (hPL/hMSCs vs rPL/rMSCs). The overarching aim was to determine the utility of hPL to foster MSC survival in preclinical rodent models. Exposure to hPL-supplemented media resulted in rMSC death, by a process attributable to heat-labile proteins, but not membrane attack complex formation. In the in vitro syngeneic model, the rodent product proved fundamentally distinct from the human product, with rPL having substantially lower growth factor content than hPL. Moreover, contrary to the positive effects of hPL on hMSC expansion, rPL did not reduce rMSC doubling time for the serum concentrations examined. When tested in vivo, hPL did not improve cell survival within hydrogel constructs through 2 weeks postimplantation. In summary, this study highlights the many facets of xenogenicity and interspecies variability that must be considered in the preclinical evaluation of hPL. Copyright © 2016 John Wiley & Sons, Ltd.

Delivery vehicle effects on bone regeneration and heterotopic ossification induced by high dose BMP-2.

Bone morphogenetic protein-2 (BMP-2), delivered on absorbable collagen sponge, is frequently used to treat bone defects. However, supraphysiological BMP-2 doses are common and often associated with complications such as heterotopic ossification and inflammation, causing pain and impaired mobility. This has prompted investigations into strategies to spatially control bone regeneration, for example growth factor delivery in appropriate scaffolds. Our objective was to investigate the spatiotemporal effects of high dose BMP-2 on bone regeneration as a function of the delivery vehicle. We hypothesized that an alginate delivery system would spatially restrict bone formation compared to a collagen sponge delivery system. In vitro, BMP-2 release was accelerated from collagen sponge compared to alginate constructs. In vivo, bone regeneration was evaluated over 12weeks in critically sized rat femoral segmental defects treated with 30µg rhBMP-2 in alginate hydrogel or collagen sponge, surrounded by perforated nanofiber meshes. Total bone volume, calculated from micro-CT reconstructions, was higher in the alginate group at 12weeks. Though bone volume within the central defect region was greater in the alginate group at 8 and 12weeks, heterotopic bone volume was similar between groups. Likewise, mechanical properties from ex vivo torsional testing were comparable between groups. Histology corroborated these findings and revealed heterotopic mineralization at 2weeks post-surgery in both groups. Overall, this study recapitulated the heterotopic ossification associated with high dose BMP-2 delivery, and demonstrated that the amount and spatial pattern of bone formation was dependent on the delivery matrix. STATEMENT OF SIGNIFICANCE: Alginate hydrogel-based BMP-2 delivery has induced better spatiotemporal bone regeneration in animals, compared to clinically used collagen sponge, at lower BMP-2 doses. Lack of clear dose-response relationships for BMP-2 vis-à-vis bone regeneration has contributed to the use of higher doses clinically. We investigated the potential of the alginate system, with comparatively favorable BMP-2 release-kinetics, to reduce heterotopic ossification and promote bone regeneration, when used with a high BMP-2 dose. While defect mineralization improved with alginate hydrogel, the initial high-release phase and likely early tissue exposure to BMP-2 appeared sufficient to induce heterotopic ossification. The characterization presented here should provide the framework for future evaluations of strategies to optimize bone formation and minimize adverse effects of high dose BMP-2 therapy.

Enhanced in vivo retention of low dose BMP-2 via heparin microparticle delivery does not accelerate bone healing in a critically sized femoral defect.

Bone morphogenetic protein-2 (BMP-2) is an osteoinductive growth factor used clinically to induce bone regeneration and fusion. Some complications associated with BMP-2 treatment have been attributed to rapid release of BMP-2 from conventional collagen scaffolds, motivating the development of tunable sustained-release strategies. We incorporated BMP-2-binding heparin microparticles (HMPs) into a hydrogel scaffold to improve spatiotemporal control of BMP-2 delivery to large bone defects. HMPs pre-loaded with BMP-2 were mixed into alginate hydrogels and compared to hydrogels containing BMP-2 alone. BMP-2 release from scaffolds in vitro, BMP-2 retention within injury sites in vivo, and bone regeneration in a critically sized femoral defect were evaluated. Compared to hydrogel delivery alone, BMP-2-loaded HMPs reduced BMP-2 release in vitro and increased early BMP-2 retention in the bone defect. BMP-2-loaded HMPs induced bone formation at both ectopic and orthotopic sites; however, the volume of induced bone was lower for defects treated with BMP-2-loaded HMPs compared to hydrogel delivery. To better understand the effect of HMPs on BMP-2 release kinetics, a computational model was developed to predict BMP-2 release from constructs in vivo. The model suggested that HMPs limited BMP-2 release into surrounding tissues, and that changing the HMP density could modulate BMP-2 release. Taken together, these experimental and computational results suggest the importance of achieving a balance of BMP-2 retention within the bone defect and BMP-2 release into surrounding soft tissues. HMP delivery of BMP-2 may provide a method of tuning BMP-2 release in vivo that can be further investigated to improve current methods of bone regeneration. STATEMENT OF SIGNIFICANCE: The development of effective biomaterials for sustained protein delivery is a crucial component of tissue engineering strategies. However, in most applications, including bone repair, the optimal balance between protein presentation in the injury site and protein release into the surrounding tissues is unknown. Herein, we introduced heparin microparticles (HMPs) into a tissue engineered construct to increase in vivo retention of bone morphogenetic protein-2 (BMP-2) and enhance healing in femoral defects. Although HMPs induced bone regeneration, no increase in bone volume was observed, leading to further experimental and computational analysis of the effect of HMP-BMP-2 interactions on protein retention and release. Ultimately, this work provides insight into designing tunable protein-material interactions and their implications for controlling BMP-2 delivery.