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.

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.

Bioprinting of exosomes: Prospects and challenges for clinical applications.

453Three-dimensional bioprinting (3DBP) is an additive manufacturing technique that has emerged as a promising strategy for the fabrication of scaffolds, which can successfully recapitulate the architectural, biochemical, and physical cues of target tissues. More importantly, 3DBP offers fine spatiotemporal control and high submicron scale resolution, which can be leveraged for the incorporation and directional gradient release of single or multiple biomimetic cues, including cell-derived exosomes (EXOs). EXOs are extracellular vesicles that originate from the endosomal compartment of various cell types, with sizes ranging from 30 to120 nm. They act as cell mediators and contain discrete cell constituents, including growth factors, cytokines, lipid moieties, nucleic acids, metabolites, and cell surface markers, depending on the cell type. Essentially, owing to their therapeutic potential, EXOs derived from mesenchymal stem cells (MSCs) have been recently investigated in several clinical trials for the treatment of various conditions, including cancer, diabetes, dry eyes, periodontitis, and acute ischemic stroke. The 3DBP strategy of EXOs is especially useful in tissue engineering and regenerative medicine applications, as tissues can be biofabricated to closely mimic the complex microarchitecture and developmental profiles of native heterogeneous tissues for restoring biological functions. Moreover, EXOs can be manipulated to carry exogenous cargo such as genes or proteins of therapeutic interest, confer multifunctional attributes, and further enhance their tissue regenerative potential. However, significant challenges, including the selection of appropriate bioink, pattern resolution, engineering-defined exosomal gradient, spatial presentation and modulation of EXO release kinetics, as well as EXO stability and storage conditions, must be addressed for the successful translation of therapeutic grade EXOs to clinical settings. In this review, we highlight the recent advances and offer future perspectives on the bioprinting of EXOs as regenerative biotherapeutics for the fabrication of complex heterogeneous tissues that are suitable for clinical transplantation.

Functionally Graded Bioactive Composites Based on Poly(vinyl alcohol) Made through Thiol-Ene Click Reaction.

Functionally graded materials (FGMs) composed of a polymer matrix embedded with calcium phosphate particles are preferred for bone tissue engineering, as they can mimic the hierarchical and gradient structure of bones. In this study, we report the design and development of a FGM based on thiolated poly(vinyl alcohol) (TPVA) and nano-hydroxyapatite (nano-HA) with graded bioactivity, cell compatibility, and degradability properties that are conducive for bone regeneration. The polymer matrix comprises crosslinked poly(vinyl alcohol) with ester and thioether linkages formed via the thiol-ene click reaction, avoiding undesired additives and byproducts. Freshly precipitated and spray-dried HA was mixed with the TPVA hydrogel, and layers of varying concentrations were cast. Upon lyophilization, the hydrogel structure yielded porous sheets of the graded composite of TPVA and nano-HA. The new FGM showed higher values of tensile strength and degradation in phosphate buffer saline (PBS) in vitro, compared to bare TPVA. The bioactive nature of the FGM was confirmed through bioactivity studies in simulated body fluid (SBF), while cytocompatibility was demonstrated with human periodontal ligament cells in vitro. Cumulatively, our results indicate that based on the composition, mechanical properties, bioactivity, and cytocompatibility, the fabricated TPVA-HA composites can find potential use as guided bone regeneration (GBR) membranes.

Human Cadaveric Donor Cornea Derived Extra Cellular Matrix Microparticles for Minimally Invasive Healing/Regeneration of Corneal Wounds.

Biological materials derived from extracellular matrix (ECM) proteins have garnered interest as their composition is very similar to that of native tissue. Herein, we report the use of human cornea derived decellularized ECM (dECM) microparticles dispersed in human fibrin sealant as an accessible therapeutic alternative for corneal anterior stromal reconstruction. dECM microparticles had good particle size distribution (≤10 µm) and retained the majority of corneal ECM components found in native tissue. Fibrin-dECM hydrogels exhibited compressive modulus of 70.83 ± 9.17 kPa matching that of native tissue, maximum burst pressure of 34.3 ± 3.7 kPa, and demonstrated a short crosslinking time of ~17 min. The fibrin-dECM hydrogels were found to be biodegradable, cytocompatible, non-mutagenic, non-sensitive, non-irritant, and supported the growth and maintained the phenotype of encapsulated human corneal stem cells (hCSCs) in vitro. In a rabbit model of anterior lamellar keratectomy, fibrin-dECM bio-adhesives promoted corneal re-epithelialization within 14 days, induced stromal tissue repair, and displayed integration with corneal tissues in vivo. Overall, our results suggest that the incorporation of cornea tissue-derived ECM microparticles in fibrin hydrogels is non-toxic, safe, and shows tremendous promise as a minimally invasive therapeutic approach for the treatment of superficial corneal epithelial wounds and anterior stromal injuries.

Fluorescence imaging of nitric oxide in living cells using o-phenylenediamine-rhodamine based polymeric nanosensors.

Direct real-time measurement of nitric oxide (NO) in living cells has proven quite challenging, owing in part to the lack of tools that are selective and sensitive to measure intracellular concentrations of NO. Herein we report the synthesis and characterization of polyvinyl alcohol (PVA) based nanosensors for fluorescence imaging of cytosolic NO using an o-phenylenediamine-rhodamine (OPD-RhB) platform. More specifically, thiol-functionalized PVA incorporating RhB conjugated OPD was disulfide crosslinked to yield NO-responsive nanosensors. The polymeric nanosensors were anionic, averaged 170 nm in hydrodynamic size, and exhibited linear increases in fluorescence intensity (FLI) to micro- and nanomolar concentrations of NO in a sodium nitroprusside (SNP) concentration-dependent manner. In the presence of SNP, the engineered nanosensors demonstrated physical stability at extracellular glutathione (GSH) conditions, while favoring NO detection at cytoplasmic GSH conditions. In addition, the PVA-based nanosensors were non-cytotoxic, cell membrane-permeable and demonstrated hydrogen peroxide-dependent FL increases upon incubation with activated synoviocytes in vitro. Most notably, NO-induced cell FLIs correlated strongly with total nitrite/nitrate content of conventional Griess assays with Pearson correlation coefficients of 0.96. Comprehensively, our results show that OPD-RhB-conjugated PVA nanosensors offer real-time imaging of NO with high sensitivity in living cells that can be employed for direct quantification of NO.

Recent Advances in Biomaterials Science and Engineering Research in India: A Minireview.

Biomedical research in health innovation and product development encompasses convergent technologies that primarily integrate biomaterials science and engineering at its core. Particularly, research in this area is instrumental for the implementation of biomedical devices (BMDs) that offer innovative solutions to help maintain and improve quality of life of patients worldwide. Despite achieving extraordinary success, implantable BMDs are still confronted with complex engineering and biological challenges that need to addressed for augmenting device performance and prolonging lifetime in vivo. Biofabrication of tissue constructs, designing novel biomaterials and employing rational biomaterial design approaches, surface engineering of implants, point of care diagnostics and micro/nano-based biosensors, smart drug delivery systems, and noninvasive imaging methodologies are among strategies exploited for improving clinical performance of implantable BMDs. In India, advances in biomedical technologies have dramatically advanced health care over the last few decades and the country is well-positioned to identify opportunities and translate emerging solutions. In this article, we attempt to capture the recent advances in biomedical research and development progressing across the country and highlight the significant research work accomplished in the areas of biomaterials science and engineering.

Bioreducible amino acid-derived polymeric nanoparticles for delivery of functional proteins.

We report the preparation of protein encapsulated amino acid derived redox-responsive nanoparticles (NPs) as effective nanocarriers for intracellular delivery of proteins. More specifically, acryloyl derivatives of lysine, ornithine, cystine and cystamine, were employed as monomers and disulfide crosslinkers for non-covalent encapsulation of model protein bovine serum albumin (BSA) and were interfacially crosslinked via free radical polymerization to form redox-responsive protein NPs. Notably, prepared NPs exhibited high protein loading content between 37 and 45%, averaged ∼400 nm in hydrodynamic size and possessed a mean surface charge of -15 mV. Furthermore, blank polymeric NPs displayed exceptional cytocompatibility with cell viability exceeding 92% at concentrations as high as 4 mg/mL, while redox-responsive protein NPs displayed glutathione (GSH)-dependent BSA release behavior in vitro. Additionally, cellular uptake studies confirmed that protein NPs entered MDA-MA-231 cells predominantly via the endocytic pathway. Upon cellular internalization, redox-responsive NPs delivered protein into cytosol of cells within 60 min demonstrating intrinsic endosomolytic characteristics and efficient protein release under cytoplasmic high GSH conditions. Most importantly, insulin analog-loaded NPs significantly increased glucose consumption in HepG2 cultures confirming protein stability and retention of protein function. Cumulatively, our approach presents a simple yet effective strategy for intracellular delivery of biologically active proteins for various biomedical applications.

Design, preparation and characterization of pH-responsive prodrug micelles with hydrolyzable anhydride linkages for controlled drug delivery.

We report a new prodrug micelle-based approach in which a model hydrophobic non-steroidal anti-inflammatory drug (NSAID), ibuprofen (Ibu), is tethered to amphiphilic methoxy polyethylene glycol-polypropylene fumarate (mPEG-PPF) diblock copolymer via hydrolytic anhydride linkages for potential controlled release applications of NSAIDs. Synthesized mPEG-PPF-Ibu polymer drug conjugates (PDCs) demonstrated high drug conjugation efficiency (∼90%) and self-assembled to form micellar nanostructures in aqueous medium with critical micelle concentrations ranging between 16 and 30µg/mL. The entrapment efficiency of Ibu in prepared PDC micelles was as high as 18% (w/w). Crosslinking of prodrug micelles with N,N'-dimethylaminoethyl methacrylate conferred pH-responsive characteristics. pH-responsive PDC micelles averaged 100nm in size at pH 7.4 and exhibited concomitant changes in size upon incubation in physiologically relevant mildly acidic conditions. Ibu release was observed to increase with increasing acidic conditions and could be controlled by varying the amount of crosslinker used. Furthermore, the prepared mPEG-PPF-based micelles demonstrated excellent cytocompatibility and cellular internalization in vitro. More importantly, PDC micelles exerted anti-inflammatory effects by significantly decreasing monosodium urate crystal-induced prostaglandin E2 levels in rabbit synoviocyte cultures in vitro. Cumulatively, our results indicate that this new prodrug micelle approach is promising for NSAID-based therapies in the treatment of arthritis and cancer.

Injectable in situ forming xylitol-PEG-based hydrogels for cell encapsulation and delivery.

Injectable in situ crosslinking hydrogels offer unique advantages over conventional prefabricated hydrogel methodologies. Herein, we synthesize poly(xylitol-co-maleate-co-PEG) (pXMP) macromers and evaluate their performance as injectable cell carriers for tissue engineering applications. The designed pXMP elastomers were non-toxic and water-soluble with viscosity values permissible for subcutaneous injectable systems. pXMP-based hydrogels prepared via free radical polymerization with acrylic acid as crosslinker possessed high crosslink density and exhibited a broad range of compressive moduli that could match the natural mechanical environment of various native tissues. The hydrogels displayed controlled degradability and exhibited gradual increase in matrix porosity upon degradation. The hydrophobic hydrogel surfaces preferentially adsorbed albumin and promoted cell adhesion and growth in vitro. Actin staining on cells cultured on thin hydrogel films revealed subconfluent cell monolayers composed of strong, adherent cells. Furthermore, fabricated 3D pXMP cell-hydrogel constructs promoted cell survival and proliferation in vitro. Cumulatively, our results demonstrate that injectable xylitol-PEG-based hydrogels possess excellent physical characteristics and exhibit exceptional cytocompatibility in vitro. Consequently, they show great promise as injectable hydrogel systems for in situ tissue repair and regeneration.

In vivo fluorescence imaging of biomaterial-associated inflammation and infection in a minimally invasive manner.

Implant-associated inflammation and bacterial infection severely limit the functional performance of medical devices and are a major cause of implant failure. Therefore, it is crucial to develop methodologies to monitor/image implant-associated aseptic inflammation and bacterial infection in a minimally invasive manner. Here, we exploited near-infrared fluorescence (NIRF) molecular probes injected locally at the implant site to perform minimally invasive, simultaneous imaging of inflammation, and infection associated with implanted polymer disks. The hydro-sulfo-Cy5 (H-s-Cy5) probe detected reactive oxygen species associated with inflammatory responses to both aseptic and biofilm-containing implants, whereas diaminocyanine sulfonate selectively detected nitric oxide associated with a biofilm on the biomaterial at acute time points (<4 days). This imaging modality also allows longitudinal monitoring because of high specificity and fast clearance rate of the fluorescent probes. Taken together, these NIRF molecular probes represent a useful tool to directly image inflammatory responses and infections associated with implanted devices for the diagnosis of device-associated inflammation and infection as well as the development of effective therapies.

PHACOS, a functionalized bacterial polyester with bactericidal activity against methicillin-resistant Staphylococcus aureus.

Biomaterial-associated infections represent a significant clinical problem, and treatment of these microbial infections is becoming troublesome due to the increasing number of antibiotic-resistant strains. Here, we report a naturally functionalized bacterial polyhydroxyalkanoate (PHACOS) with antibacterial properties. We demonstrate that PHACOS selectively and efficiently inhibits the growth of methicillin-resistant Staphylococcus aureus (MRSA) both in vitro and in vivo. This ability has been ascribed to the functionalized side chains containing thioester groups. Significantly less (3.2-fold) biofilm formation of S. aureus was detected on PHACOS compared to biofilms formed on control poly(3-hydroxyoctanoate-co-hydroxyhexanoate) and poly(ethylene terephthalate), but no differences were observed in bacterial adhesion among these polymers. PHACOS elicited minimal cytotoxic and inflammatory effects on murine macrophages and supported normal fibroblast adhesion. In vivo fluorescence imaging demonstrated minimal inflammation and excellent antibacterial activity for PHACOS compared to controls in an in vivo model of implant-associated infection. Additionally, reductions in neutrophils and macrophages in the vicinity of sterile PHACOS compared to sterile PHO implant were observed by immunohistochemistry. Moreover, a similar percentage of inflammatory cells was found in the tissue surrounding sterile PHACOS and S. aureus pre-colonized PHACOS implants, and these levels were significantly lower than S. aureus pre-colonized control polymers. These findings support a contact active surface mode of antibacterial action for PHACOS and establish this functionalized polyhydroxyalkanoate as an infection-resistant biomaterial.

Diverse mediators modulate the chloride ion fluxes that drive lacrimal fluid production.

PURPOSE: To learn whether locally expressed and systemic mediators might modulate the cholinergically induced transepithelial Cl(-) fluxes that underlie lacrimal fluid production. METHODS: Reconstituted epithelial monolayers were exposed to a submaximal dose of the muscarinic agonist, carbachol (CCh), or to one of several paracrine mediators for 18 hours, then acutely stimulated with an optimal dose of CCh. Secretory Cl(-) fluxes were assessed as negative short-circuit currents (ISC). RESULTS: Exposure to IL-6 at concentrations of 1 and 10 ng/mL and IL-1ß at 10 ng/mL significantly decreased CCh-induced Cl(-) secretion. Prolactin decreased CCh-induced Cl(-) secretion, but the extent of the decrease diminished as the prolactin concentration increased from 20 to 200 ng/mL. CCh, 10 µM, prevented CCh, 100 µM, from eliciting Cl(-) secretion. Exposure to histamine, 10 mM, prevented formation of confluent monolayers. Exposure to histamine, 1 mM, decreased CCh-induced Cl(-) secretion, whereas exposure to 5-HT, 1 mM, potentiated CCh-induced Cl(-) secretion. CONCLUSIONS: Chronic exposure to inflammatory cytokines may significantly impair cholinergically induced lacrimal fluid production. Concentrations of prolactin within the high range of normal values also may impair fluid production, but this effect is reversed at levels associated with pregnancy. Autonomic neurotransmitters and paracrine mediators that signal through different G protein-coupled receptors appear to exert varying influences, which range from complete suppression to potentiation of cholinergically induced fluid production. Thus, some hormones and paracrine mediators may impair secretion in apparently homeostatic glands as well as diseased glands, whereas mediators produced by certain immune cell infiltrates may actually enhance fluid formation.

Minimally invasive, longitudinal monitoring of biomaterial-associated inflammation by fluorescence imaging.

Implant-associated inflammation is a major cause for the reduced performance/lifetime and failure of numerous medical devices. Therefore, the ability to non-invasively and quantitatively monitor implant-associated inflammation is critically important. Here we show that implant-associated inflammation can be imaged via fluorescence imaging using near-infrared hydrocyanine dyes delivered either locally or intravenously in living mice. This imaging strategy allowed quantitative longitudinal monitoring of inflammation by detecting reactive oxygen species (ROS) released by inflammatory cells in response to implanted poly(ethylene terephthalate) (PET) disks or injected poly (lactic-co-glycolic acid) (PLGA) microparticles, and exhibited a strong correlation to conventional analysis of inflammation. Furthermore, modulation of inflammatory responses via controlled release of the anti-inflammatory agent dexamethasone was detected using this sensitive imaging approach. Thus, hydrocyanine-based fluorescence imaging of ROS could serve as a surrogate measure for monitoring implant-associated inflammation as well as evaluating the efficacy of therapeutic approaches to modulate host responses to implanted medical devices.

Distinct dacryoadenitides autoadoptively transferred to rabbits by different subpopulations of lymphocytes activated ex vivo.

PURPOSE: To test whether CD4+ T cells proliferate in mixed cell reactions with autologous lacrimal gland (LG) acinar cells and whether these cells can autoadoptively transfer disease. METHODS: Purified acinar cells were gamma irradiated and cocultured with peripheral blood lymphocytes. Activated CD4+ T cells were sorted by fluorescence-activated cell sorting (FACS). Unfractionated activated peripheral blood lymphocytes (UF), CD4+-enriched and CD4+-depleted T cells from an autologous mixed cell reaction were injected into the donor rabbit's remaining LG. After 4 weeks, ocular examinations were performed, and the rabbits were euthanized; LGs were removed for histopathology, immunohistochemistry, and real-time reverse transcription-polymerase chain reaction studies. RESULTS: CD4 T cells increased in the autologous mixed cell reaction from 20% to 80%. Tear production decreased in the induced disease/UF (ID/UF) group and declined even more in the ID/CD4+-enriched group. Tear breakup times decreased and rose bengal staining increased in all groups. All LGs exhibited significant histopathology and increased messenger RNAs for tumor necrosis factor α. The ID/UF group exhibited the largest increases of CD4+ and rabbit T-lymphocyte antigen-positive cells. The ID/CD4+-enriched group contained fewer infiltrating CD4 cells but more eosinophils, severely altered acinar morphology, and increased fibrosis. LG of the ID/CD4+-depleted group exhibited large increases of CD18, major histocompatibility complex II, and CD4+ cells. Messenger RNAs for interleukin 2, interleukin 4, and CD4+ increased in the ID/CD4+-enriched group compared with the CD4+-depleted group. CONCLUSIONS: Autoreactive CD4+ effector cells activated ex vivo and autoadoptively transferred, caused what seems to be a distinct dacryoadenitis. The CD4+-depleted cell fraction also contained pathogenic effector cells capable of inducing disease.

Adeno-associated virus-mediated IL-10 gene transfer suppresses lacrimal gland immunopathology in a rabbit model of autoimmune dacryoadenitis.

PURPOSE: To evaluate the effect of adeno-associated virus (AAV) vector-mediated viral (v)IL-10 gene expression on lacrimal gland (LG) immunopathology and ocular surface disease in a rabbit model of induced autoimmune dacryoadenitis (ID). METHODS: Autologous peripheral blood lymphocytes, activated in a mixed-cell reaction when cocultured with purified rabbit lacrimal epithelial cells, induce a Sjögren's-like autoimmune dacryoadenitis when injected directly back into the donor animal's inferior LG. Four weeks after disease induction, AAV vector expressing the vIL-10 gene under control of a tetracycline-inducible promoter was injected into the inferior LG of the treatment group (ID/Rx), and doxycycline was fed orally to induce transgene expression. The ID group serving as control also received doxycycline. All LGs were removed 16 weeks after disease induction. RESULTS: Clinical symptoms showed overall improvement in the ID/Rx group compared with the ID group. Histopathologic examination of the ID group's LG revealed scattered large lymphocytic foci and areas of altered or distorted acini, whereas the ID/Rx group had scattered small lymphocytic foci. The number of CD18(+) cells was almost fivefold lower in the ID/Rx group than in the ID group. Although the total number of RTLA(+) cells did not differ between the groups, the CD4/CD8 ratio was 16-fold smaller in the ID/Rx group. CONCLUSIONS: Animals with experimentally induced autoimmune dacryoadenitis appeared to benefit from AAV-mediated vIL-10 gene transfer therapy. Quantitative immunohistochemical analysis suggested that the therapy might not have been simply immunosuppressive but rather supported the induction of CD8(+) regulatory cells.

Long-term topical cyclosporine treatment improves tear production and reduces keratoconjunctivitis in rabbits with induced autoimmune dacryoadenitis.

PURPOSE: To use a rabbit model of induced autoimmune dacryoadenitis to evaluate the efficacy of topical ophthalmic cyclosporine A (CsA). METHODS: Autoimmune dacryoadenitis was induced by injecting autologous peripheral blood lymphocytes, which had been activated in a mixed cell reaction with acinar cells isolated from one inferior lacrimal gland (LG), back into the donor animal's remaining inferior LG. Schirmer's test, tear breakup time, and rose Bengal staining were assessed. Animals with established disease were treated topically with either CsA or Endura twice daily for 5 months. RESULTS: Without treatment tear production and tear stability were abnormal for 6 months, and clear signs of ocular surface defects were evident. Severe immune cell infiltration was observed in the LG. Long-term CsA treatment increased tear production only slightly, but the severity of LG histopathology decreased noticeably. CD4(+) T-cell infiltration of the LG was decreased and infiltration by MHC class II-expressing cells was also decreased. For the Endura-treated group tear production did not improve, rose Bengal scores remained high, and histopathology showed infiltration comparable to the untreated group, but by the end of the study the tear breakup time did improve. CONCLUSIONS: The rabbit model of autoimmune dacryoadenitis had signs of chronic dry eye disease 6 months after induction of disease. Tear production improved slightly with CsA treatment and CD4(+) T-cell infiltration decreased significantly in the LG. This suggests that some Sjögren's patients may benefit from long-term CsA treatment.

Microporous poly(L-lactic acid) membranes fabricated by polyethylene glycol solvent-cast/particulate leaching technique.

With the eventual goal of developing a tissue-engineered tear secretory system, we found that primary lacrimal gland acinar cells grown on solid poly(L-lactic acid) (PLLA) supports expressed the best histiotypic morphology. However, to be able to perform vectorial transport functions, epithelia must be supported by a permeable substratum. In the present study, we describe the use of a solvent-cast/particulate leaching technique to fabricate microporous PLLA membranes (mpPLLAm) from PLLA/polyethylene glycol blends. Scanning electron microscopy revealed pores on both the air-cured ( approximately 4 microm) and glass-cured sides (<2 microm) of the mpPLLAm. Diffusion studies were performed with mpPLLAm fabricated from 57.1% PLLA/42.9% polyethylene glycol blends to confirm the presence of channelized pores. The data reveal that glucose, L-tryptophan, and dextran (a high molecular weight glucose polymer) readily permeate mpPLLAm. Diffusion of the immunoglobulin G through the mpPLLAm decreased with time, suggesting the possible adsorption and occlusion of the pores. Cells cultured on the mpPLLAm (57.1/42.9 wt%) grew to subconfluent monolayers but retained histiotypic morphological and physiological characteristics of lacrimal acinar cells in vivo. Our results suggest that mpPLLAm fabricated using this technique may be useful as a scaffold for a bioartificial lacrimal gland device.

Cytopathology and exocrine dysfunction induced in ex vivo rabbit lacrimal gland acinar cell models by chronic exposure to histamine or serotonin.

PURPOSE: Lacrimal immunohistopathology has diverse clinical presentations, suggesting that inflammatory mediators exert diverse influences. Chronic exposure to agonistic acetylcholine receptor autoantibodies has been studied previously; the present work addressed mediators that signal through other G protein-coupled receptors. METHODS: Acinus-like structures and reconstituted acinar epithelial monolayers from rabbit lacrimal glands were exposed to varying concentrations of histamine or 5-hydroxytryptamine (5-HT) for 20 hours. Net and vectorial beta-hexosaminidase secretion, cytosolic Ca(2+) (Ca(i)) elevation, apical recruitment of p150(Glued), actin microfilament meshwork organization, and ultrastructure were assessed. RESULTS: Histamine and 5-HT acutely stimulated beta-hexosaminidase secretion at lower, but not higher, concentrations. Neither of them acutely elevated Ca(i) levels. Both recruited p150(Glued) at concentrations that failed to induce secretion. Chronic exposure to 10 mM histamine inhibited carbachol (CCh)-induced beta-hexosaminidase secretion and prevented the formation of continuous monolayers; 1 mM 5-HT partially inhibited secretion at the apical medium. Neither altered secretion to the basal medium. Chronic exposure to histamine or 5-HT partially decreased CCh induced Ca(i) elevations and p150(Glued) recruitment, even at concentrations that did not inhibit secretion. Both expanded acinar lumina and thickened microfilament meshworks, and both caused homotypic fusion of secretory vesicles and formation of aqueous vacuoles in the apical and basal cytoplasm. Chronic exposure to forskolin, which activates adenylyl cyclase, induced similar cytopathologic changes but impaired secretion modestly and only at the highest concentration tested. CONCLUSIONS: Inflammatory mediators that signal through G protein-coupled receptors cause acinar cell cytopathology and dose-dependent reductions of CCh-induced beta-hexosaminidase secretion. Although agonistic acetylcholine receptor autoantibodies may cause pervasive functional quiescence, inflammatory mediators may cause varying degrees of exocrine dysfunction.

Transepithelial bioelectrical properties of rabbit acinar cell monolayers on polyester membrane scaffolds.

In our quest to develop a tissue-engineered tear secretory system, we have tried to demonstrate active transepithelial ion fluxes across rabbit lacrimal acinar cell monolayers on polyester membrane scaffolds to evaluate the bioelectrical properties of the cultured cells. Purified lacrimal gland acinar cells were seeded onto polyester membrane inserts and cultured to confluency. Morphological properties of the cell monolayers were evaluated by transmission electron microscopy and immunofluorescence staining for Na(+),K(+)-ATPase and the tight junction-associated protein occludin. Sections revealed cell monolayers with well-maintained epithelial cell polarity, i.e., presence of apical (AP) secretory granules, microvilli, and junctional complexes. Na(+),K(+)-ATPase was localized on both the basal-lateral and apical plasma membranes. The presence of tight cell junctions was demonstrated by a positive circumferential stain for occludin. Bioelectrical properties of the cell monolayers were studied in Ussing chambers under short-circuit conditions. Active ion fluxes were evaluated by inhibiting the short-circuit current (I(sc)) with a Na(+),K(+)-ATPase inhibitor, ouabain (100 microM; basal-lateral, BL), and under Cl(-)-free buffer conditions after carbachol stimulation (CCh; 100 microM). The directional apical secretion of Cl(-) was demonstrated through pharmacological analysis, using amiloride (1 mM; BL) and bumetanide (0.1 mM; BL), respectively. Regulated protein secretion was evaluated by measuring the beta-hexosaminidase catalytic activity in the AP culture medium in response to 100 microM basal CCh. In summary, rabbit lacrimal acinar cell monolayers generate a Cl(-)-dependent, ouabain-sensitive AP --> BL I(sc) in response to CCh, consistent with current models for Na(+)-dependent Cl(-) secretion.