Cytocompatibility study of NHLBI primary reference materials using human endothelial cells.

Four different materials, low density polyethylene (LDPE), polydimethylsiloxane (PDMS), polyvinylcholoride (PVC) and cellulose, were selected by the Devices and Technology Branch of the National Heart Lung and Blood Institute (NHLBI) as primary reference materials for blood contacting. Among the wide variety of tests proposed to assess hemocompatibility of short-term blood contacting catheters, it was desirable to rule out whether these materials could release toxics for vascular cells of the physiological environment. Thus, the cytocompatibility of these materials have been checked towards human umbilical vein endothelial cells: the method used avoids direct contact between cells and materials but evaluates the effect of possible toxic substances leached from materials. These substances were obtained under defined conditions according to a standard. The results show that the extracts of cellulose and LDPE provoke an important cytotoxic effect on the endothelial cell cultures, while the extracts of PDMS and PVC allow the obtention of endothelial cell lining of the reference surface, with a correct global metabolic activity and the intracellular presence of von Willebrand factor.     

Hemocompatibility, biocompatibility, inflammatory and in vivo studies of primary reference materials low-density polyethylene and polydimethylsiloxane: a review

In 1984, low-density polyethylene (LDPE) and polymethylsiloxane (PDMS), two primary reference materials (PRM), were made available by the National Heart, Lung, and Blood Institute (NHLBI) as discriminatory tools for the validation of standardized and novel in vitro and in vivo tests in the evaluation of biomaterials. This article reviews the results and conclusions obtained by several studies investigating the hemocompatibility, in vitro biocompatibility, inflammatory response, and in vivo tissue reactions of these two reference materials. Variable results obtained with LDPE and PDMS in ex vivo hemocompatibility studies were attributed to the type of animal model used, the flow velocity of the circulating blood, the time of exposure, and the methodology used to measure blood cell adhesion or activation at the surface of the materials. In contrast, both the LDPE and PDMS appeared to be suitable reference materials when used in in vitro biocompatibility, inflammatory response, and in vivo studies. However, caution must be taken when interpreting the results, because gamma sterilization of these two materials as well as their origin (for example PDMS) are two critically important factors. In conclusion, we see a definite need for standardized hemocompatible parameters and better high-quality hemocompatibility studies on PRM. This review also suggests other materials as potential PRM candidates, namely, Biomer and Intramedic polyethylene.     

In vivo leucocyte interactions with the NHLBI-DTB primary reference materials: Polyethylene and silica-free polydimethylsiloxane

In vivo leucocyte interactions with the NHLBI-DTB primary reference materials, low density polyethylene (LDPE) and silica-free polydimethylsiloxane (PDMS), were qualitatively and quantitatively characterized using a cage implant system over a 21 d implantation period. Scanning electron microscopy (SEM) and cytochemical staining procedures were utilized to observe the cellular events occurring at the leucocyte/ biomaterial interface. The results showed that more cells adhered to the PDMS surface than the LDPE surface at days 4 and 7. The differential analysis revealed that mononuclear cells, presumably macrophages, preferentially adhered to both polymer surfaces. By day 21, there were more very large (> 20 nuclei per cell) foreign body giant cells (FBGCs) present on the PDMS surface than the LDPE surface. The phagocytic capabilities of the adhered cells, including the FBGCs, decreased to a greater extent on the PDMS surface, corresponding to the earlier and more extensive spreading of these cells observed in the morphological analysis.     

The interaction of blood components with PDMS (polydimethylsiloxane) and LDPE (low-density polyethylene) in a baboon ex vivo arteriovenous shunt model

The interaction of 111Indium-labeled platelets, and other blood components with the luminal surface of polydimethylsiloxane (PDMS) and low-density polyethylene (LDPE) was determined using an ex vivo arteriovenous shunt in the baboon. Both PDMS and LDPE showed little platelet accumulation at either high (200 cc/min) flow rates. PDMS accumulated more platelets at low than at high flow. When the surfaces of PDMS and LDPE were examined under scanning electron microscopy after 2.5 h of flow in the shunt circuit, red and white blood cells were attached. Platelets appeared to be confined to patchy areas covered by a fibrinlike network. The low platelet reactivity of LDPE and PDMS suggests their potential use as coatings for conventional, more platelet reactive vascular graft materials.     

Nanocomposite Ti/hydrocarbon plasma polymer films from reactive magnetron sputtering as growth support for osteoblast-like and endothelial cells

Nanocomposite Ti/hydrocarbon plasma polymer (Ti/ppCH) films were deposited by DC magnetron sputtering of titanium target in n-hexane, argon, or a mixture of these two gases. The resultant films were heterogeneous, with inorganic regions of nanometer scale distributed within a plasma polymer matrix. The titanium content was controlled by adjusting the argon/n-hexane ratio in the working gas. In the pure n-hexane atmosphere, the Ti concentration was found to be below 1 at %, whereas in pure argon it reached 20 at %, as measured by Rutherford backscattering spectroscopy and elastic recoil detection analysis (RBS/ERDA). A high level of titanium oxidation is detected with TiO(2), substoichiometric titania, and titanium carbide, composing an inorganic phase of the composite films. In addition, high hydrogen content is detected in films rich with titanium. Ti-deficient and Ti-rich films proved equally good substrates for adhesion and growth of cultured human osteoblast-like MG 63 cells. In these cells, the population densities on days 1, 3, and 7 after seeding, spreading area on day 1, formation of talin-containing focal adhesion plaques as well as concentrations of talin and osteocalcin (per mg of protein) were comparable to the values obtained in cells on the reference cell culture materials, represented by microscopic glass coverslips or a polystyrene dish. An interesting finding was made when the Ti/ppCH films were seeded with calf pulmonary artery endothelial cells of the line CPAE. The cell population densities, the spreading area and also the concentration of von Willebrand factor, a marker of endothelial cell maturation, were significantly higher on Ti-rich than on Ti-deficient films. On Ti-rich films, these parameters were also higher or similar in comparison with the reference cell culture materials. Thus, both types of films could be used for coating bone implants, of which the Ti-rich film remains effective in enhancing the endothelialization of blood contacting artificial materials.     

New strategies for in vivo tissue engineering by mimicry of homing factors for self-endothelialisation of blood contacting materials

For years intensive research has been done to endothelialise vascular prostheses with autologous endothelial cells before implantation in patients. However, this procedure is extremely time-, labor- and cost-intensive and can be realized only in very few clinical cases. The discovery of circulating endothelial progenitor cells (EPCs) in 1997 brought new perspectives for the endothelialisation of blood contacting materials. Coating of synthetic graft surfaces with capture molecules for circulating EPCs mimics a pro-homing substrate for fishing out EPCs directly from the bloodstream after implantation. These cells with high proliferation potential can cover the graft with non-thrombogenic endothelium which maintains optimal haemostasis and minimize the risk of restenosis. In this review, different concepts are discussed to capture circulating EPCs on synthetic vascular grafts after implantation. We hypothesize that in vivo self-endothelialisation of blood contacting materials by homing factor-mimetic capture molecules for EPCs may bring revolutionary new perspectives towards future clinical application of stem cell and tissue engineering strategies.     

In vitro evaluation of phosphonylated low-density polyethylene for vascular applications

The use of catheters for vascular applications is often complicated by the development of friction between the catheter material and the vessel wall, which leads to endothelial cell removal and intimal lesions. Phosphonylation, a chemical surface treatment, has been proposed as a means of increasing the hydrophilicity of low-density polyethylene (LDPE), a commonly used catheter material, in efforts to impart lubricity to the material and reduce vascular tissue damage. In an in vitro tribological study, phosphonylated LDPE produced a lower coefficient of friction and allowed greater retention of endothelial cells on vessels as compared to untreated LDPE when the materials were reciprocated against normal porcine aorta. Chemical characterizations of the LDPE before and after friction testing involving Fourier transform infrared and energy-dispersive X-ray (EDX) confirmed the phosphorus content on phosphonylated LDPE. Election spectroscopy for chemical analysis (ESCA) and atomic force micrscope (AFM) analyses verified that proteins initially adsorb to both the phosphonylated and untreated LDPE surfaces and that the proteins interfere with water to lubricate the surfaces. However, with repeated friction, proteins are removed from the surface and hydrophilicity, as imparted by phosphonylation, becomes a principal factor in the lubrication process.     

Festschrift in honor of the 65th birthday of Dr John L. Brash. Part 2

Adsorption of fibrinogen from buffer as a single protein and from plasma to four materials has been studied. The two NIH-NHLBI primary reference standards, filler free polydimethylsiloxane and low density polyethylene, were used along with polyvinylchloride and cellulose materials supplied by the IUPAC Working Party. The materials were examined in both film and tubing form, except for polydimethylsiloxane which was studied only in tubing form. Adsorption was measured at room temperature using ¹²⁵I-Iabelled fibrinogen. The order of adsorbed amounts in the single protein experiments was found to be: cellulose < PVC < PE = PDMS. Apparent adsorption affinities are in the same order. In plasma, all surfaces except cellulose showed maxima in adsorption as a function of plasma concentration after 5 min contact. This is indicative of initial adsorption followed by displacement of fibrinogen (the Vroman effect). Cellulose showed very low adsorption of fibrinogen from plasma. The Vroman maxima were more pronounced on the tubing samples than on the films, and, as for the single protein experiments, adsorption was found to be less on tubing than on film samples. A tentative interpretation of the Vroman effect data suggests that the order of procoagulant activity of the materials may be: PDMS = PE < PVC < cellulose.     

Improving bacterial cellulose for blood vessel replacement: Functionalization with a chimeric protein containing a cellulose-binding module and an adhesion peptide

Chimeric proteins containing a cellulose-binding module (CBM) and an adhesion peptide (RGD or GRGDY) were produced and used to improve the adhesion of human microvascular endothelial cells (HMEC) to bacterial cellulose (BC). The effect of these proteins on the HMEC–BC interaction was studied. The results obtained demonstrated that recombinant proteins containing adhesion sequences were able to significantly increase the attachment of HMEC to BC surfaces, especially the RGD sequence. The images obtained by scanning electron microscopy showed that the cells on the RGD-treated BC present a more elongated morphology 48 h after cell seeding. The results also showed that RGD decreased the in-growth of HMEC cells through the BC and stimulated the early formation of cord-like structures by these endothelial cells. Thus, the use of recombinant proteins containing a CBM domain, with high affinity and specificity for cellulose surfaces allows control of the interaction of this material with cells. CBM may be combined with virtually any biologically active protein for the modification of cellulose-based materials, for in vitro or in vivo applications.     

A hemangioendothelioma-derived cell line: its use as a model for the study of endothelial cell biology

The purpose of our research was to develop a stable murine cell line that could serve as a general model for the study of microvascular endothelial cells. To do so we have characterized an endothelial cell line originally derived from a mouse hemangioendothelioma. Endothelioma cells synthesize angiotensin-converting enzyme, express surface receptors for acetylated low density lipoprotein, produce thrombospondin and show intracellular staining with an antibody to von Willebrand antigen. The response of endothelioma cells to endothelial cell mitogens, known angiogenesis-inducing factors, extracellular matrix substrate (Matrigel), and various cytokines was measured. Endothelioma cells show an increased proliferative response to extracts from hypothalamus but only marginally to recombinant fibroblast growth factor or purified endothelial cell growth factor. Rearrangement of cells forming tube-like connections were induced by Matrigel. The chemokinetic responses generally correlated with angiogenesis-inducing properties of the test substances. The results support the conclusion that the endothelioma cell line maintains characteristic endothelial cell properties and behaves in vitro in a manner similar to microvascular endothelial cells.     

Characterization of the inflammatory response to biomaterials using a rodent air pouch model

Using a rodent air pouch, the inflammatory responses to biomaterials with distinct physical properties and chemical compositions were compared. The polymers examined were expanded poly(tetrafluoroethylene) (ePTFE), silicone, low-density polyethylene (LDPE), poly(L-lactic acid) (PLLA), poly(desaminotyrosyl-tyrosine ethyl carbonate) [poly(DTE carbonate)], and poly(desaminotyrosyl-tyrosine benzyl carbonate) [poly(DTBzl carbonate)]. We found that implantation of disks (4.5-4.8 mm) of these materials into rodent air pouches for 2 days had no effect on the number or type of cells recovered relative to sham controls. With each of the materials, macrophages were the predominant cell type identified (60-75%), followed by granulocytes (20-25%) and lymphocytes (10%). Implantation of poly(DTE carbonate), ePTFE, LDPE, or poly(DTBzl carbonate) into the pouches for 2 days caused an increase in release of superoxide anion by the pouch cells. Cells from pouches containing poly(DTE carbonate) also released more hydrogen peroxide and were more phagocytic. In contrast, PLLA and silicone had no effect on the functional activity of cells recovered from the pouches. Prolonging the implantation time of poly(DTE carbonate) or PLLA to 7 days did not alter the number or type of cells isolated from the pouches. However, cells from pouches containing poly(DTE carbonate) for 7 days continued to produce increased quantities of superoxide anion relative to sham control pouch cells. These results suggest that the air pouch model is a highly sensitive method and therefore useful for evaluating the functional responses of inflammatory cells to biomaterials.     

Directed type IV collagen self-assembly on hydroxylated PTFE

A method for the creation of a type IV collagen (CNIV) scaffold on polytetrafluoroethylene (PTFE) for endothelial cell attachment is described. This mimic for the basal lamina can be used in the seeding and retention of endothelial cells for blood contacting devices. The CNIV-PTFE production technique can be defined as three processes: (i) creation of a reactive superacidic/ionic PTFE surface with retained hydrophobic characteristics; (ii) activation of this surface via covalent attachment of N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide (EDC); and (iii) conjugation of the EDC with human CNIV resulting in the covalent binding of protein to the PTFE surface. This article demonstrates exciting new results showing that a reaction of CNIV to this particular surface results in a unique matrix assembly of CNIV scaffolds similar to those found in the basal lamina. This assembly is concentration-dependant, occurring in a narrow window around 0.435 microM. Following the fabrication of the CNIV matrix assembly, porcine aortic endothelial cells (PAEC) were seeded onto this material. Results described in this article demonstrate that the PAEC subsequently aligned with the direction of shear, filled voids created by dead or detached cells, and divided during the 6 h of experimentation. Under static conditions, cells remained viable for 1 week of testing. This was not observed with PAEC attached to glass with adsorbed Vitrogen. In summary, this article describes a novel biotechnological breakthrough that enables the creation of stable endothelial cell monolayers useful for fabricating blood contacting devices.     

Mechanism of the induction of angiogenesis by human neoplastic lymphoid tissue: studies employing bovine aortic endothelial cells in vitro

Extracts of human neoplastic tissue (three Hodgkin's lymphomas; three gliomas and one liposarcoma) previously shown to induce angiogenesis in vivo did not stimulate the proliferation of bovine aortic endothelial cell monolayers when tested in vitro. Proliferation was induced by coculture of endothelial cells with human or animal macrophages or with supernatants derived from these cells. However, angiogenic extracts were chemotactic both for guinea pig peritoneal macrophages and human mononuclear cells in vitro. These results imply that tumour extracts act indirectly to induce angiogenesis in vivo via their effect on host macrophages.     

Probing of peripheral blood mononuclear cells anchoring on TNF-alpha challenged-vascular endothelia in an <Emphasis Type="Italic">in vitro</Emphasis> model of the retinal microvascular

Retinopathy is a complication of diabetes that affects the eyes; it stems from damage to the microvasculature of the retina and eventually compromises vision. The diagnosis of retinopathy is difficult to make because there are no early symptoms or warning signs. Dysfunction of the retina’s microvascular networks is believed to be associated with inflammatory cytokines and tumor necrosis factor alpha (TNF-α). To investigate the effect of these cytokines, such as TNF-α, a polydimethylsiloxane (PDMS)/glass hydride microfluidic device reflecting the physiological structure of the retina’s microvasculature was developed. In this model, the bifurcations and tortuosity of branch vessels were based on photographs of the fundus and an endothelial cell layer (EA.hy926 cells) were reconstructed within the microfluidic network. The adhesion, spreading, and growth of cells was ensured by optimizing the conditions for cell seeding and perfusion. Fluorescent staining was used to visualize the cytoskeleton and measurement of the nitric oxide (NO) level proved that the endothelial EA.hy926 cells had spread in the direction of flow perfusion system, forming artificial vascular networks. The endothelial layer was further challenged by TNF-α perfusion. Cytokine treatment increased the anchoring of peripheral blood mononuclear cells (PBMCs) on the endothelial layer. The microfluidic device developed in this study provides a low-cost platform reflecting the physiological structures of the retina’s microvasculature. It is anticipated that this device will be useful in evaluating the diseased retina as well as in drug screening.     

不同血液透析膜材料治疗重度胰腺炎的应用价值

BACKGROUND: The hemodialysis treatment for severe pancreatitis patients can effectively remove all kinds of toxic substances, but it is unclear whether there are differences in the removal effects of different dialysis membrane materials. OBJECTIVE: To explore the clinical application value of hemodialysis membrane materials in the treatment of severe pancreatitis. METHODS: Totally 67 patients with severe pancreatitis were enrolled, including 38 males and 29 females, aged 34-81 years, and all underwent continuous bedside hemodialysis within 72 hours of onset. Among them, 32 patients selected polysulfone membrane, and the other 35 patients selected cellulose acetate membrane. Dialysis was done 5 hours once, three times a week. All the patients in these two groups were subjected to stable dialysis for 3 weeks. After 3 weeks, the recovery time required for the levels of serum amylase and white blood cell count in the two groups, treatment efficiency, liver and kidney clinical indicators and levels of inflammatory cytokines were observed and detected. RESULTS AND CONCLUSION: There were no significant differences in the recovery time required for serum amylase and white blood cell count, the incidence of adverse reactions and the effective rate of treatment between polysulfone membrane and cellulose acetate membrane groups. The acute physiology and chronic health scores, the levels of serum creatinine, triglycerides, blood amylase, alanine aminotransferase, interleukin-6, C-reactive protein and tumor necrosis factor-α after treatment in these two groups were significantly lower than those before treatment (P < 0.05). The blood O2 pressure was significantly higher than that before treatment (P < 0.05), but there were no significant differences in the above indicators between these two groups. These results demonstrate that the dialysis effects of polysulfone and cellulose acetate membranes are equivalent, both of which can effectively remove the harmful toxins in the body.      

Bartonella bacilliformis stimulates endothelial cells in vitro and is angiogenic in vivo.

Bartonellosis, a biphasic disease caused by motile intracellular bacteria, produces in its tissue phase a characteristic dermal eruption (Verruga peruana) resulting from a pronounced endothelial cell proliferation. Bacteria are found in the interstitium and within the cytoplasm of endothelial cells (Rocha-Lima inclusion). The aim of this study was to determine if Bartonella bacilliformis produce a substance(s) that might be responsible for the vascular proliferation seen in the Verruga. This was assessed in an in vitro system using human endothelial cells and measuring proliferation as well as production of tissue type plasminogen activator after exposure to the endothelial cultures to B. bacilliformis extracts. Our results indicate that B. bacilliformis possess an activity that stimulates endothelial cell proliferation up to three times that of control. The factor(s) is specific for endothelial cells, heat sensitive, larger than 12 to 14 kd, not enhanced by heparin, has no affinity for heparin, and is precipitated by 45% ammonium sulfate. In addition, the B. bacilliformis extracts stimulate production of t-PA antigen in a concentration-dependent fashion. This activity is also heat sensitive and not lost after dialysis (12 to 14 kd). B. bacilliformis extracts, however, do not increase the production of plasminogen activator inhibitor. It was also determined that B. bacilliformis extracts stimulate the formation of new blood vessels in an in vivo model for angiogenesis. These results describe a bacterial factor(s) that stimulates two important steps in the development of new blood vessels in vitro, as well as the formation of new blood vessels in vivo. Determining the mechanism of action, combined with a complete characterization of this factor(s), may help in understanding the pathogenesis not only of the Verruga and angiogenesis in general but also the recently described Cat-Scratch-associated epithelioid hemangiomas in patients with AIDS and Kaposi sarcoma.     

Pathological changes in cerebral arteries following experimental subarachnoid hemorrhage: role of blood platelets

The role of blood platelets in producing early intimal changes in cerebral arteries following subarachnoid hemorrhage (SAH) was examined by using 18 cats. Experimental SAH was produced by a rupture of the proximal portion of the right middle cerebral artery. Following SAH, the scanning electron microscope revealed that structural alterations in the intimal layer of major cerebral arteries occurred as early as 2 hours and became more severe by 48 hours. Vascular alterations, which were predominantly detected in the ruptured vessel, consisted of endothelial cell corrugation, detachment, crater formation, intimal adhesion of platelets and red blood cells, intimal thrombi, and reendothelialization. When cats were pretreated prior to SAH with an anti-platelet-aggregating agent, OKY-1581, the intimal blood elements and thrombi were clearly reduced, and reendothelialization was not observed. However, endothelial cell changes in the OKY-1581-treated group were very similar to those occurring in the nontreated group. While these results suggest that bioactive substances contained within blood platelets, such as growth factors, serotonin, and norepinephrine, have little effect on producing endothelial cell injury, platelets may be important in the initiation of reendothelialization following vessel injury.     

Endothelialized Networks with a Vascular Geometry in Microfabricated Poly(dimethyl siloxane)

One key challenge in regenerating vital organs is the survival of transplanted cells. To meet their metabolic requirements, transport by diffusion is insufficient, and a convective pathway, i.e., a vasculature, is required. Our laboratory pioneered the concept of engineering a vasculature using microfabrication in silicon and Pyrex. Here we report the extension of this concept and the development of a methodology to create an endothelialized network with a vascular geometry in a biocompatible polymer, poly(dimethyl siloxane) (PDMS). High-resolution PDMS templates were produced by replica-molding from micromachined silicon wafers. Closed channels were formed by bonding the patterned PDMS templates to flat PDMS sheets using an oxygen plasma. Human microvascular endothelial cells (HMEC-1) were cultured for 2 weeks in PDMS networks under dynamic flow. The HMEC-1 cells proliferated well in these confined geometries (channel widths ranging from 35 mum to 5 mm) and became confluent after four days. The HMEC-1 cells lined the channels as a monolayer and expressed markers for CD31 and von Willebrand factor (vWF). These results demonstrate that endothelial cells can be cultured in confined geometries, which is an important step towards developing an in vitro vasculature for tissue-engineered organs.     

Poly(dimethyl siloxane) surface modification with biosurfactants isolated from probiotic strains

Depending on the final application envisaged for a given biomaterial, many surfaces must be modified before use. The material performance in a biological environment is mainly mediated by its surface properties that can be improved using suitable modification methods. The aim of this work was to coat poly(dimethyl siloxane) (PDMS) surfaces with biosurfactants (BSs) and to evaluate how these compounds affect the PDMS surface properties. BSs isolated from four probiotic strains (Lactococcus lactis, Lactobacillus paracasei, Streptococcus thermophilus A, and Streptococcus thermophilus B) were used. Bare PDMS and PDMS coated with BSs were characterized by contact angle measurements, infrared spectroscopy (ATR-FTIR), X-ray photoelectron spectroscopy (XPS), and atomic force microscopy (AFM). The influence of the surface modifications on the materials blood compatibility was studied through thrombosis and hemolysis assays. The cytotoxicity of these materials was tested against rat peritoneal macrophages. AFM results demonstrated the successful coating of the surfaces. Also, by contact angle measurements, an increase of the coated surfaces hydrophilicity was seen. Furthermore, XPS analysis indicated a decrease of the silicon content at the surface, and ATR-FTIR results showed the presence of BS characteristic groups as a consequence of the modification. All the studied materials revealed no toxicity and were found to be nonhemolytic. The proposed approach for the modification of PDMS surfaces was found to be effective and opens new possibilities for the application of these surfaces in the biomedical field.     

Dermatophagoides extract-treated confluent type II epithelial cells (cA549) and human lung mesenchymal cell growth.

BACKGROUND: Chronic severe persistent asthma is associated with damaged epithelial cells with discontinuous tight junctions that contribute to dysregulated fibroblast and endothelial cell (mesenchymal) growth. Dermatophagoides species-derived proteases have been shown to cause damage to epithelial cell tight junctions. OBJECTIVE: To determine whether Dermatophagoides species can stimulate confluent A549 (cA549), a cell type with discontinuous tight junctions that approximate differentiated type II cells, to undergo altered growth and secrete putative soluble factors that affect the growth of human lung fibroblasts and microvascular endothelial cells. METHODS: Dialyzed Dermatophagoides pteronyssinus or Dermatophagoides farinae extracts (0, 300, 600, and 1000 AU/mL) were cultured with and without cA549 in serum-free media for 24 hours. After changes in cA549 growth were recorded, conditioned media from extracts with cA549 (CM) and without cA549 (control media [CTLM]) were transferred to fibroblasts and endothelial cells for 24 hours. Fibroblast and endothelial cell growth responses to CM and CTLM were observed and measured. RESULTS: All conditions showed greater than 95% cell viability. Confluent A549 showed dose-dependent growth changes characterized by increased aggregation when incubated with 300, 600, and 1000 AU/mL of D pteronyssinus in serum-free media relative to control. The CM, but not the CTLM, induced dose-dependent aggregation by fibroblasts and endothelial cells. Fibroblasts also showed decreased adhesion when incubated with CM. Dermatophagoides farinae-treated cA549 showed similar but weaker results. The use of serum, boiled CM, or boiled extract inhibited these findings. CONCLUSIONS: Dialyzed Dermatophagoides species extracts altered cA549 growth and stimulated the secretion of factors that dysregulate mesenchymal cell growth in vitro.