In vitro inflammatory multi-cellular model of osteoarthritis.

Objective: Osteoarthritis (OA) is a chronic joint disease, with limited treatment options, characterized by inflammation and matrix degradation, and resulting in severe pain or disability. Progressive inflammatory interaction among key cell types, including chondrocytes and macrophages, leads to a cascade of intra- and inter-cellular events which culminate in OA induction. In order to investigate these interactions, we developed a multi-cellular in vitro OA model, to characterize OA progression, and identify and evaluate potential OA therapeutics in response to mediators representing graded levels of inflammatory severity. Methods: We compared macrophages, chondrocytes and their co-culture responses to "low" Interleukin-1 (IL-1) or "high" IL-1/tumor necrosis factor (IL-1/TNF) levels of inflammation. We also investigated response changes following the administration of dexamethasone (DEX) or mesenchymal stromal cell (MSC) treatment via a combination of gene expression and secretory changes, reflecting not only inflammation, but also chondrocyte function. Results: Inflamed chondrocytes presented an osteoarthritic-like phenotype characterized by high gene expression of pro-inflammatory cytokines and chemokines, up-regulation of ECM degrading proteases, and down-regulation of chondrogenic genes. Our results indicate that while MSC treatment attenuates macrophage inflammation directly, it does not reduce chondrocyte inflammatory responses, unless macrophages are present as well. DEX however, can directly attenuate chondrocyte inflammation. Conclusions: Our results highlight the importance of considering multi-cellular interactions when studying complex systems such as the articular joint. In addition, our approach, using a panel of both inflammatory and chondrocyte functional genes, provides a more comprehensive approach to investigate disease biomarkers, and responses to treatment.

Chitosan-polygalacturonic acid complex dressing improves diabetic wound healing and hair growth in diabetic mice.

Chronic skin wounds decrease the quality of life of millions of diabetic patients worldwide. Chitosan has previously been shown to possess hemostatic properties, decrease inflammation, promote fibroblast proliferation, and hair growth. We developed a relatively low-cost polyelectrolyte complex (PEC) film dressing made of chitosan and polygalacturonic acid and tested it for its ability to accelerate diabetic wound healing. Genetically diabetic male mice were shaved on the dorsum, and one day later a 1 cm diameter full-thickness excisional wound was created. The PEC film was applied immediately after wounding and left in place for 14 days. Controls consisted of wounds treated with a fibrin gel. Wounds covered with the PEC film had closed completely by post-wounding day 42, while untreated wounds were only half-way closed. Histological analysis of wounds confirmed that PEC-treated wounds had fully re-epithelialized, while control wounds lacked a continuous epidermis at the wound center. We also observed that the area of skin under the PEC film experienced much more rapid hair growth. Histologically, there were significantly more hair follicles around the scar area (p < 0.05) in the PEC-treated group as compared to the control group. Thus, chitosan-polygalacturonic acid PEC films can accelerate both wound healing and hair growth in diabetic mice, and should be further investigated as a potential future treatment for diabetic chronic wounds.

Autofluorescence of blood and its application in biomedical and clinical research.

Autofluorescence of blood has been explored as a label free approach for detection of cell types, as well as for diagnosis and detection of infection, cancer, and other diseases. Although blood autofluorescence is used to indicate the presence of several physiological abnormalities with high sensitivity, it often lacks disease specificity due to use of a limited number of fluorophores in the detection of several abnormal conditions. In addition, the measurement of autofluorescence is sensitive to the type of sample, sample preparation, and spectroscopy method used for the measurement. Therefore, while current blood autofluorescence detection approaches may not be suitable for primary clinical diagnosis, it certainly has tremendous potential in developing methods for large scale screening that can identify high risk groups for further diagnosis using highly specific diagnostic tests. This review discusses the source of blood autofluorescence, the role of spectroscopy methods, and various applications that have used autofluorescence of blood, to explore the potential of blood autofluorescence in biomedical research and clinical applications.

HSymM-guided engineering of the immunodominant p53 transactivation domain putative peptide antigen for improved binding to its anti-p53 monoclonal antibody.

A novel engineering strategy to improve autoantibody detection with peptide fragments derived from the parent antigen is presented. The model system studied was the binding of the putative p53 TAD peptide antigen (residues 46-55) to its cognate anti-p53 antibody, ab28. Each engineered peptide contained the full decapeptide epitope and differed only in the flanking regions. Since minimal structural information was available to guide the design, a simple epitope:paratope binding model was applied. The Hidden Symmetry Model, which we recently reported, was used to guide peptide design and estimate per-residue contributions to interaction free energy as a function of added C- and N-terminal flanking peptides. Twenty-four peptide constructs were designed, synthesized, and assessed for binding affinity to ab28 by surface plasmon resonance, and a subset of these peptides were evaluated in a simulated immunoassay for limit of detection. Many peptides exhibited over 200-fold enhancements in binding affinity and improved limits of detection. The epitope was reevaluated and is proposed to be the undecapeptide corresponding to residues 45-55. HSymM calculated binding free energy and experimental data were found to be in good agreement (R2 > 0.75).

Anti-inflammatory effects of haptoglobin on LPS-stimulated macrophages: Role of HMGB1 signaling and implications in chronic wound healing.

Nonhealing wounds possess elevated numbers of pro-inflammatory M1 macrophages, which fail to transition to anti-inflammatory M2 phenotypes that promote healing. Hemoglobin (Hb) and haptoglobin (Hp) proteins, when complexed (Hb-Hp), can elicit M2-like macrophages through the heme oxygenase-1 (HO-1) pathway. Despite the fact that nonhealing wounds are chronically inflamed, previous studies have focused on non-inflammatory systems, and do not thoroughly compare the effects of complexed vs individual proteins. We aimed to investigate the effect of Hb/Hp treatments on macrophage phenotype in an inflammatory, lipopolysaccharide (LPS)-stimulated environment, similar to chronic wounds. Human M1 macrophages were cultured in vitro and stimulated with LPS. Concurrently, Hp, Hb, or Hb-Hp complexes were delivered. The next day, 27 proteins related to inflammation were measured in the supernatants. Hp treatment decreased a majority of inflammatory factors, Hb increased many, and Hb-Hp had intermediate trends, indicating that Hp attenuated overall inflammation to the greatest extent. From this data, Ingenuity Pathway Analysis software identified high motility group box 1 (HMGB1) as a key canonical pathway-strongly down-regulated from Hp, strongly up-regulated from Hb, and slightly activated from Hb-Hp. HMGB1 measurements in macrophage supernatants confirmed this trend. In vivo results in diabetic mice with biopsy punch wounds demonstrated accelerated wound closure with Hp treatment, and delayed wound closure with Hb treatment. This work specifically studied Hb/Hp effects on macrophages in a highly inflammatory environment relevant to chronic wound healing. Results show that Hp-and not Hb-Hp, which is known to be superior in noninflammatory conditions-reduces inflammation in LPS-stimulated macrophages, and HMGB1 signaling is also implicated. Overall, Hp treatment on M1 macrophages in vitro reduced the inflammatory secretion profile, and also exhibited benefits in in silico and in vivo wound-healing models.

A controlled release bupivacaine-alginate construct: Effect on chondrocyte hypertrophy conversion.

Objective: Osteoarthritis is a degenerative disease of the joint, affecting over 30 million people in the US1. A key characteristic of OA is chondrocyte hypertrophy, characterized by chondrocyte changes to a more rounded and osteoblastic phenotype, characterized by increased IL-6 and IL-8 secretion2. While there are no cures for OA, treatments focus on mitigating pain and inflammation, the two main symptoms of OA. However, the analgesics, NSAIDS and corticosteroids commonly used, do not target regeneration and have negative side effects. Local anesthetics (LA) can be used as a pain management alternative but are usually short lasting and therefore, not suited for chronic conditions such as OA. Our engineered sustained release local anesthetic construct successfully delivers bupivacaine for an extended period of time3-5. This study is designed to evaluate the effect of the LA system on chondrocytes in an inflammatory OA-like environment. Design: Chondrocytes were cultured with bolus, liposomal, or construct LA and either untreated or treated with TNF-α and IL-1α for 24 hrs, 48 hrs, or 96 hrs. Chondrocyte viability, interleukin-8 (IL-8), interleukin-6 (IL-6), collagenase activity and proteoglycan deposition were assessed. Results: In the presence of the engineered construct, the chondrocytes retained viability and regenerative function. Moreover, the construct allowed for higher initial doses to be used, which promoted more regeneration and decreased inflammation without compromising cellular viability. Conclusions: The construct promotes a less hypertrophic chondrocyte environment while promoting a more anti-inflammatory environment. These two factors are consistent with a less OA progressive environment when using the engineered construct, compared to bolus LA.

Dynamin and reverse-mode sodium calcium exchanger blockade confers neuroprotection from diffuse axonal injury.

Mild traumatic brain injury (mTBI) is a frequently overlooked public health concern that is difficult to diagnose and treat. Diffuse axonal injury (DAI) is a common mTBI neuropathology in which axonal shearing and stretching induces breakdown of the cytoskeleton, impaired axonal trafficking, axonal degeneration, and cognitive dysfunction. DAI is becoming recognized as a principal neuropathology of mTBI with supporting evidence from animal model, human pathology, and neuroimaging studies. As mitochondrial dysfunction and calcium overload are critical steps in secondary brain and axonal injury, we investigated changes in protein expression of potential targets following mTBI using an in vivo controlled cortical impact model. We show upregulated expression of sodium calcium exchanger1 (NCX1) in the hippocampus and cortex at distinct time points post-mTBI. Expression of dynamin-related protein1 (Drp1), a GTPase responsible for regulation of mitochondrial fission, also changes differently post-injury in the hippocampus and cortex. Using an in vitro model of DAI previously reported by our group, we tested whether pharmacological inhibition of NCX1 by SN-6 and of dynamin1, dynamin2, and Drp1 by dynasore mitigates secondary damage. Dynasore and SN-6 attenuate stretch injury-induced swelling of axonal varicosities and mitochondrial fragmentation. In addition, we show that dynasore, but not SN-6, protects against H2O2-induced damage in an organotypic oxidative stress model. As there is currently no standard treatment to mitigate cell damage induced by mTBI and DAI, this work highlights two potential therapeutic targets for treatment of DAI in multiple models of mTBI and DAI.

Alginate encapsulation for bupivacaine delivery and mesenchymal stromal cell immunomodulatory cotherapy.

PURPOSE: Mesenchymal stromal cells (MSCs) are used to treat various inflammatory conditions. In parallel, to mitigate pain associated with inflammation, analgesics or opioids are prescribed, often with significant side effects. Local anesthetics (LAs) offer a promising alternative to these medications. However, their short duration and negative effects on anti-inflammatory MSCs have limited their therapeutic effectiveness. To mitigate these negative effects and to move toward developing a cotherapy, we engineered a sustained release bupivacaine alginate-liposomal construct that enables up to 4 days of LA release. By encapsulating MSC in alginate (eMSC), we demonstrate that we can further increase drug concentration to clinically relevant levels, without compromising eMSC viability or anti-inflammatory function. MATERIALS AND METHODS: MSCs were freely cultured or encapsulated in alginate microspheres ± TNFα/IFN-γ and were left untreated or dosed with bolus, liposomal, or construct bupivacaine. After 24, 48, and 96 hours, the profiles were assessed to quantify secretory function associated with LA-MSC interactions. To approximate LA exposure over time, a MATLAB model was generated. RESULTS: eMSCs secrete similar levels of IL-6 and prostaglandin E2 (PGE2) regardless of LA modality, whereas free MSCs secrete larger amounts of IL-6 and lower amounts of anti-inflammatory PGE2. Modeling the system indicated that higher doses of LA can be used in conjunction with eMSC while retaining eMSC viability and function. In general, eMSC treated with higher doses of LA secreted similar or higher levels of immunomodulatory cytokines. CONCLUSION: eMSCs, but not free MSC, are protected from LA, regardless of LA modality. Increasing the LA concentration may promote longer and stronger pain mitigation while the protected eMSCs secrete similar, if not higher, immunomodulatory cytokine levels. Therefore, we have developed an approach, using eMSC and the LA construct that can potentially be used to reduce pain as well as improve MSC anti-inflammatory function.

Macrophage modulation by polymerized hemoglobins: Potential as a wound-healing therapy.

Chronic skin wounds are hypoxic and are stalled in a pro-inflammatory state. Hemoglobin (Hb)-based oxygen carriers have shown potential in increasing oxygen delivery to aid wound healing. Macrophages also take up Hb, thus altering their phenotype and the regulation of inflammation. Herein, we compared the effect of Hb and polymerized Hbs (PolyHbs) on the phenotype of human macrophages. Macrophages were incubated with Hb or different forms of PolyHbs, and the inflammatory secretion profile was analyzed. PolyHbs were produced by polymerizing Hb in the relaxed (R) or tense (T) quaternary state and by varying the molar ratio of the glutaraldehyde crosslinking agent to Hb. Hb decreased the secretion of most measured factors. PolyHb treatment led to generally similar secretion profiles; however, Hb had more similar trends to R-state PolyHb. Ingenuity pathway analysis predicted positive outcomes in wound healing and angiogenesis for T-state PolyHb prepared with a 30:1 (glutaraldehyde:Hb) polymerization ratio. When tested in diabetic mouse wounds, T-state PolyHb resulted in the greatest epidermal thickness and vascular endothelial CD31 staining. Thus, the effects of PolyHb on macrophages are affected by the polymerization ratio and the quaternary state, and T-state PolyHb yields secretion profiles that are most beneficial in wound healing.

Label-free dynamic segmentation and morphological analysis of subcellular optical scatterers.

Imaging without fluorescent protein labels or dyes presents significant advantages for studying living cells without confounding staining artifacts and with minimal sample preparation. Here, we combine label-free optical scatter imaging with digital segmentation and processing to create dynamic subcellular masks, which highlight significantly scattering objects within the cells' cytoplasms. The technique is tested by quantifying organelle morphology and redistribution during cell injury induced by calcium overload. Objects within the subcellular mask are first analyzed individually. We show that the objects' aspect ratio and degree of orientation ("orientedness") decrease in response to calcium overload, while they remain unchanged in untreated control cells. These changes are concurrent with mitochondrial fission and rounding observed by fluorescence, and are consistent with our previously published data demonstrating scattering changes associated with mitochondrial rounding during calcium injury. In addition, we show that the magnitude of the textural features associated with the spatial distribution of the masked objects' orientedness values, changes by more than 30% in the calcium-treated cells compared with no change or changes of less than 10% in untreated controls, reflecting dynamic changes in the overall spatial distribution and arrangement of subcellular scatterers in response to injury. Taken together, our results suggest that our method successfully provides label-free morphological signatures associated with cellular injury. Thus, we propose that dynamically segmenting and analyzing the morphology and organizational patterns of subcellular scatterers as a function of time can be utilized to quantify changes in a given cellular condition or state.

Microfluidic flow cytometry: The role of microfabrication methodologies, performance and functional specification.

Flow cytometry is an invaluable tool utilized in modern biomedical research and clinical applications requiring high throughput, high resolution particle analysis for cytometric characterization and/or sorting of cells and particles as well as for analyzing results from immunocytometric assays. In recent years, research has focused on developing microfluidic flow cytometers with the motivation of creating smaller, less expensive, simpler, and more autonomous alternatives to conventional flow cytometers. These devices could ideally be highly portable, easy to operate without extensive user training, and utilized for research purposes and/or point-of-care diagnostics especially in limited resource facilities or locations requiring on-site analyses. However, designing a device that fulfills the criteria of high throughput analysis, automation and portability, while not sacrificing performance is not a trivial matter. This review intends to present the current state of the field and provide considerations for further improvement by focusing on the key design components of microfluidic flow cytometers. The recent innovations in particle focusing and detection strategies are detailed and compared. This review outlines performance matrix parameters of flow cytometers that are interdependent with each other, suggesting trade offs in selection based on the requirements of the applications. The ongoing contribution of microfluidics demonstrates that it is a viable technology to advance the current state of flow cytometry and develop automated, easy to operate and cost-effective flow cytometers.

Microfluidic platforms for the study of neuronal injury in vitro.

Traumatic brain injury (TBI) affects 5.3 million people in the United States, and there are 12,500 new cases of spinal cord injury (SCI) every year. There is yet a significant need for in vitro models of TBI and SCI in order to understand the biological mechanisms underlying central nervous system (CNS) injury and to identify and test therapeutics to aid in recovery from neuronal injuries. While TBI or SCI studies have been aided with traditional in vivo and in vitro models, the innate limitations in specificity of injury, isolation of neuronal regions, and reproducibility of these models can decrease their usefulness in examining the neurobiology of injury. Microfluidic devices provide several advantages over traditional methods by allowing researchers to (1) examine the effect of injury on specific neural components, (2) fluidically isolate neuronal regions to examine specific effects on subcellular components, and (3) reproducibly create a variety of injuries to model TBI and SCI. These microfluidic devices are adaptable for modeling a wide range of injuries, and in this review, we will examine different methodologies and models recently utilized to examine neuronal injury. Specifically, we will examine vacuum-assisted axotomy, physical injury, chemical injury, and laser-based axotomy. Finally, we will discuss the benefits and downsides to each type of injury model and discuss how researchers can use these parameters to pick a particular microfluidic device to model CNS injury.

Alginate-liposomal construct for bupivacaine delivery and MSC function regulation.

Mesenchymal stromal cell (MSC) therapies have become potential treatment options for multiple ailments and traumatic injuries. In the clinical setting, MSC are likely to be co-administered with local anesthetics (LA) which have been shown to have dose- and potency-dependent detrimental effects on the viability and function of cells. We previously developed and characterized a sustained-release LA delivery formulation comprised of alginate-encapsulated liposomal bupivacaine. The current studies were designed to evaluate the effect of this formulation on the secretion of three key MSC regulatory molecules, interleukin 6 (IL-6), prostaglandin E2 (PGE2), and transforming growth factor-beta 1 (TGF-ß1). MSCs were treated with several bupivacaine formulations-bolus, liposome, or alginate-liposome construct (engineered construct)-in the presence or absence of inflammatory stimulus to stimulate an injured tissue environment. Our results indicated that compared to bolus or liposomal bupivacaine, the engineered construct preserved or promoted MSC anti-inflammatory PGE2 secretion; however, the engineered construct did not increase TGF-ß1 secretion. Bupivacaine release profile analyses indicated that mode of drug delivery controlled the LA concentration over time and pathway analysis identified several shared and cytokine-specific molecular mediators for IL-6, PGE2, and TGF-ß1 which could explain differential MSC secretion responses in the presence of bupivacaine. Collectively, these studies support the potential utility of alginate-encapsulated LA constructs for anti-inflammatory cell therapy co-administration and indicate that mode of local anesthetic delivery can significantly alter MSC secretome function.

The growing role of precision and personalized medicine for cancer treatment.

Cancer is a devastating disease that takes the lives of hundreds of thousands of people every year. Due to disease heterogeneity, standard treatments, such as chemotherapy or radiation, are effective in only a subset of the patient population. Tumors can have different underlying genetic causes and may express different proteins in one patient versus another. This inherent variability of cancer lends itself to the growing field of precision and personalized medicine (PPM). There are many ongoing efforts to acquire PPM data in order to characterize molecular differences between tumors. Some PPM products are already available to link these differences to an effective drug. It is clear that PPM cancer treatments can result in immense patient benefits, and companies and regulatory agencies have begun to recognize this. However, broader changes to the healthcare and insurance systems must be addressed if PPM is to become part of standard cancer care.

Prostaglandin E2 Produced by Alginate-Encapsulated Mesenchymal Stromal Cells Modulates the Astrocyte Inflammatory Response.

Astroglia are well known for their role in propagating secondary injury following brain trauma. Modulation of this injury cascade, including inflammation, is essential to repair and recovery. Mesenchymal stromal cells (MSCs) have been demonstrated as trophic mediators in several models of secondary CNS injury, however, there has been varied success with the use of direct implantation due to a failure to persist at the injury site. To achieve sustained therapeutic benefit, we have encapsulated MSCs in alginate microspheres and evaluated the ability of these encapsulated MSCs to attenuate neuro-inflammation. In this study, astroglial cultures were administered lipopolysaccharide (LPS) to induce inflammation and immediately co-cultured with encapsulated or monolayer human MSCs. Cultures were assayed for the pro-inflammatory cytokine tumor necrosis factor alpha (TNF-α) produced by astroglia, MSC-produced prostaglandin E2, and expression of neurotrophin-associated genes. We found that encapsulated MSCs significantly reduced TNF-α produced by LPS-stimulated astrocytes, more effectively than monolayer MSCs, and this enhanced benefit commences earlier than that of monolayer MSCs. Furthermore, in support of previous findings, encapsulated MSCs constitutively produced high levels of PGE2, while monolayer MSCs required the presence of inflammatory stimuli to induce PGE2 production. The early, constitutive presence of PGE2 significantly reduced astrocyte-produced TNF-α, while delayed administration had no effect. Finally, MSC-produced PGE2 was not only capable of modulating inflammation, but appears to have an additional role in stimulating astrocyte neurotrophin production. Overall, these results support the enhanced benefit of encapsulated MSC treatment, both in modulating the inflammatory response and providing neuroprotection.

The effect of local anesthetic on pro-inflammatory macrophage modulation by mesenchymal stromal cells.

Administering local anesthetics (LAs) peri- and post-operatively aims to prevent or mitigate pain in surgical procedures and after tissue injury in cases of osteoarthritis (OA) and other degenerative diseases. Innovative tissue protective and reparative therapeutic interventions such as mesenchymal stromal cells (MSCs) are likely to be exposed to co-administered drugs such as LAs. Therefore, it is important to determine how this exposure affects the therapeutic functions of MSCs and other cells in their target microenvironment. In these studies, we measured the effect of LAs, lidocaine and bupivacaine, on macrophage viability and pro-inflammatory secretion. We also examined their effect on modulation of the macrophage pro-inflammatory phenotype in an in vitro co-culture system with MSCs, by quantifying macrophage tumor necrosis factor (TNF)-α secretion and MSC prostaglandin E2 (PGE2) production. Our studies indicate that both LAs directly attenuated macrophage TNF-α secretion, without significantly affecting viability, in a concentration- and potency-dependent manner. LA-mediated attenuation of macrophage TNF-α was sustained in co-culture with MSCs, but MSCs did not further enhance this anti-inflammatory effect. Concentration- and potency-dependent reductions in macrophage TNF-α were concurrent with decreased PGE2 levels in the co-cultures further indicating MSC-independent macrophage attenuation. MSC functional recovery from LA exposure was assessed by pre-treating MSCs with LAs prior to co-culture with macrophages. Both MSC attenuation of TNF-α and PGE2 secretion were impaired by pre-exposure to the more potent bupivacaine and high dose of lidocaine in a concentration-dependent manner. Therefore, LAs can affect anti-inflammatory function by both directly attenuating macrophage inflammation and MSC secretion and possibly by altering the local microenvironment which can secondarily reduce MSC function. Furthermore, the LA effect on MSC function may persist even after LA removal.

Metabolic Flux Distribution during Defatting of Steatotic Human Hepatoma (HepG2) Cells.

Methods that rapidly decrease fat in steatotic hepatocytes may be helpful to recover severely fatty livers for transplantation. Defatting kinetics are highly dependent upon the extracellular medium composition; however, the pathways involved are poorly understood. Steatosis was induced in human hepatoma cells (HepG2) by exposure to high levels of free fatty acids, followed by defatting using plain medium containing no fatty acids, or medium supplemented with a cocktail of defatting agents previously described before. We measured the levels of 28 extracellular metabolites and intracellular triglyceride, and fed the data into a steady-state mass balance model to estimate strictly intracellular fluxes. We found that during defatting, triglyceride content decreased, while beta-oxidation, the tricarboxylic acid cycle, and the urea cycle increased. These fluxes were augmented by defatting agents, and even more so by hyperoxic conditions. In all defatting conditions, the rate of extracellular glucose uptake/release was very small compared to the internal supply from glycogenolysis, and glycolysis remained highly active. Thus, in steatotic HepG2 cells, glycolysis and fatty acid oxidation may co-exist. Together, these pathways generate reducing equivalents that are supplied to mitochondrial oxidative phosphorylation.

Donor variability among anti-inflammatory pre-activated mesenchymal stromal cells.

Therapeutic mesenchymal stromal cells (MSCs) are attractive in part due to their immunomodulatory properties, achieved by their paracrine secretion of factors including prostaglandin E2 (PGE2). Despite promising pre-clinical data, demonstrating clinical efficacy has proven difficult. The current studies were designed to develop approaches to pre-induce desired functions from naïve MSCs and examine MSC donor variability, two factors contributing to this disconnect. MSCs from six human donors were pre-activated with interleukin 1 beta (IL-1ß) at a concentration and duration identified as optimal or interferon gamma (IFN-γ) as a comparator. Their secretion of PGE2 after pre-activation and secondary exposure to pro-inflammatory molecules was measured. Modulation of tumor necrosis factor alpha (TNF-α) secretion from M1 pro-inflammatory macrophages by co-cultured pre-activated MSCs was also measured. Our results indicated that pre-activation of MSCs with IL-1ß resulted in upregulated PGE2 secretion post exposure. Pre-activation with IL-1ß or IFN-γ resulted in higher sensitivity to induction by secondary stimuli compared to no pre-activation. While IL-1ß pre-activation led to enhanced MSC-mediated attenuation of macrophage TNF-α secretion, IFN-γ pre-activation resulted in enhanced TNF-α secretion. Donor variability was noted in PGE2 secretion and upregulation and the level of improved or impaired macrophage modulation.

Effect of Local Anesthetics on Human Mesenchymal Stromal Cell Secretion.

Anti-fibrotic and tissue regenerative mesenchymal stromal cell (MSC) properties are largely mediated by secreted cytokines and growth factors. MSCs are implanted to augment joint cartilage replacement and to treat diabetic ulcers and burn injuries simultaneously with local anesthetics, which reduce pain. However, the effect of anesthetics on therapeutic human MSC secretory function has not been evaluated. In order to assess the effect of local anesthetics on the MSC secretome, a panel of four anesthetics with different potencies - lidocaine, procaine, ropivacaine and bupivacaine - was evaluated. Since injured tissues secrete inflammatory cytokines, the effects of anesthetics on MSCs stimulated with tumor necrosis factor (TNF)-α and interferon (IFN)-γ were also measured. Dose dependent and anesthesia specific effects on cell viability, post exposure proliferation and secretory function were quantified using alamar blue reduction and immunoassays, respectively. Computational pathway analysis was performed to identify upstream regulators and molecular pathways likely associated with the effects of these chemicals on the MSC secretome. Our results indicated while neither lidocaine nor procaine greatly reduced unstimulated cell viability, ropivacaine and bupivacaine induced dose dependent viability decreases. This pattern was exaggerated in the simulated inflammatory environment. The reversibility of these effects after withdrawal of the anesthetics was attenuated for TNF-α/IFN-γ-stimulated MSCs exposed to ropivacaine and bupivacaine. In addition, secretome analysis indicated that constitutive secretion changes were clearly affected by both anesthetic alone and anesthetic plus TNFα/IFNγ cell stimulation, but the secretory pattern was drug specific and did not necessarily coincide with viability changes. Pathway analysis identified different intracellular regulators for stimulated and unstimulated MSCs. Within these groups, ropivacaine and bupivacaine appeared to act on MSCs similarly via the same regulatory mechanisms. Given the variable effect of local anesthetics on MSC viability and function, these studies underscore the need to evaluate MSC in the presence of medications, such as anesthetics, that are likely to accompany cell implantation.