Tunable Keratin Hydrogels for Controlled Erosion and Growth Factor Delivery.

Tunable erosion of polymeric materials is an important aspect of tissue engineering for reasons that include cell infiltration, controlled release of therapeutic agents, and ultimately to tissue healing. In general, the biological response to proteinaceous polymeric hydrogels is favorable (e.g., minimal inflammatory response). However, unlike synthetic polymers, achieving tunable erosion with natural materials is a challenge. Keratins are a class of intermediate filament proteins that can be obtained from several sources, including human hair, and have gained increasing levels of use in tissue engineering applications. An important characteristic of keratin proteins is the presence of a large number of cysteine residues. Two classes of keratins with different chemical properties can be obtained by varying the extraction techniques: (1) keratose by oxidative extraction and (2) kerateine by reductive extraction. Cysteine residues of keratose are "capped" by sulfonic acid and are unable to form covalent cross-links upon hydration, whereas cysteine residues of kerateine remain as sulfhydryl groups and spontaneously form covalent disulfide cross-links. Here, we describe a straightforward approach to fabricate keratin hydrogels with tunable rates of erosion by mixing keratose and kerateine. SEM imaging and mechanical testing of freeze-dried materials showed similar pore diameters and compressive moduli, respectively, for each keratose-kerateine mixture formulation (∼1200 kPa for freeze-dried materials and ∼1.5 kPa for hydrogels). However, the elastic modulus (G') determined by rheology varied in proportion with the keratose-kerateine ratios, as did the rate of hydrogel erosion and the release rate of thiol from the hydrogels. The variation in keratose-kerateine ratios also led to tunable control over release rates of recombinant human insulin-like growth factor 1.

Ciprofloxacin-loaded keratin hydrogels reduce infection and support healing in a porcine partial-thickness thermal burn.

Infection is a leading cause of morbidity and mortality in burn patients. Current therapies include silver-based creams and dressings, which display limited antimicrobial effectiveness and impair healing. The need exists for a topical, point-of-injury antibiotic treatment that provides sustained antimicrobial activity without impeding wound repair. Fitting this description are keratin-based hydrogels, which are fully biocompatible and support the slow-release of antibiotics. Here we develop a porcine model of an infected partial-thickness burn to test the effects of ciprofloxacin-loaded keratin hydrogels on infection and wound healing. Partial-thickness burns were inoculated with either Pseudomonas aeruginosa or Methicillin-resistant Staphylococcus aureus, resulting in infections that persisted for >2 weeks that exceeded 10(5) and 10(6) cfu per gram of tissue, respectively. Compared to silver sulfadiazine, ciprofloxacin-loaded keratin hydrogel treatment significantly reduced the amount of P. aeruginosa and S. aureus in the burn by >99% on days 4, 7, 11, and 15 postinjury. Further, burns treated with ciprofloxacin-loaded keratin hydrogels exhibited similar healing patterns as uninfected burns with regards to reepithelialization, macrophage recruitment, and collagen deposition and remodeling. The ability of keratin hydrogels to deliver antibiotics to fight infection and support healing of partial-thickness burns make them a strong candidate as a first-line burn therapy.

Subcutaneous Anti-inflammatory Therapies to Prevent Burn Progression in a Swine Model of Contact Burn Injury.

INTRODUCTION: If left untreated, burn injuries can deepen or progress in depth within the first 72 hours after injury as a result of increased wound inflammation, subsequently worsening healing outcomes. This can be especially detrimental to warfighters who are constrained to resource-limited environments with delayed evacuation times to higher roles of care and more effective treatment. Preventing this burn progression at the point of injury has the potential to improve healing outcomes but requires a field-deployable therapy and delivery system. Subcutaneous therapies known to treat inflammation delivered local to the wound site may prove to be one such avenue for success. MATERIALS AND METHODS: Seven Yorkshire-cross swine received partial-thickness burn injuries using a previously established contact burn model. Each animal received one of the seven therapies: (1) saline, (2) heparin, (3) ibuprofen, (4) erythropoietin, (5) resolvin, (6) rapamycin, and (7) placental extract, all of which are either currently employed or are experimental in field use and indicated to treat inflammation. Treatments were delivered subcutaneously on the day of injury and 24 hours post-injury to simulate a prolonged field care scenario, before potential evacuation. Animals and wound development were observed for 28 days before euthanasia. Throughout the course of the study, wounds were observed macroscopically via non-invasive imaging. Histological analyses provided the critical metric of burn progression. Treatment success criteria were designated as the ability to prevent burn progression past 80% of the dermal depth in two of the three treated wounds, a clinically relevant metric of burn progression. RESULTS: It was determined that the applied model successfully created reproducible partial-thickness burn injuries in this porcine study. No significant differences with regard to lateral wound size or the rate of lateral wound closure were observed in any treatments. Several treatments including resolvin, rapamycin, ibuprofen, and erythropoietin successfully reduced burn progression to less than 80% of the dermal depth in two of the three wounds, 24 hours after injury. CONCLUSIONS: This report employs an established model of porcine contact burn injury in order to test the ability of local subcutaneous delivery of therapeutics to prevent burn progression at the point of injury, via what is believed to be the inhibition of inflammation. Several treatments successfully prevented burn progression to a full-thickness injury, potentially improving wound healing outcomes in a simulated battlefield scenario. Subcutaneously administered therapies combating burn-induced inflammation at the point of injury may serve as a field-deployable treatment modality to improve warfighter recovery and return to duty.

Halofuginone infused keratin hydrogel attenuates adhesions in a rodent cecal abrasion model.

BACKGROUND: Postoperative adhesion formation continues to be a significant surgical complication, and methods for preventing abdominopelvic adhesions remain limited. Halofuginone (HF) is a type-1 collagen synthesis inhibitor and may enhance the effects of a physical barrier in preventing adhesion formation. We evaluated the effectiveness of a HF infused keratin hydrogel on preventing adhesions in a rat cecal abrasion model. MATERIAL AND METHODS: Laparotomy and standardized cecal abrasion was performed on 58 retired-breeder Sprague Dawley female rats to induce intra-abdominal adhesions. Rats were randomized to: no treatment; Interceed absorbable adhesion barrier; keratin hydrogel alone; or keratin hydrogel infused with 22 µg/mL of HF. Necropsies were performed at postop d-14 to assess the extent and tenacity of adhesions and grade histologic inflammation and fibrosis using a standard scoring system. Serum, liver, kidneys, and lungs were harvested to evaluate tissue HF concentrations. Protein and drug elution curves were generated to assess the release of HF from the hydrogel. RESULTS: Treatment with Keratin-HF hydrogel resulted in significantly fewer abdominal adhesions than any other treatment, and significantly less dense adhesions compared with Interceed or keratin hydrogel alone. Subset histologic analysis did not reveal qualitative differences. HF was undetectable in serum and kidneys, and detected at negligible concentrations in liver and lungs. Keratin-HF hydrogel drug release in phosphate-buffered solution (PBS) was sustained over 7 d and correlated with keratin protein degradation. CONCLUSIONS: Keratin-HF hydrogel is a novel therapeutic agent that may provide a better method for preventing the development of postoperative adhesions using a combined physical barrier and pharmacologic approached.

Combination therapy using three novel prolactin receptor antagonist-based fusion proteins effectively inhibits tumor recurrence and metastasis in HER2/neu transgenic mice.

Previously, prolactin receptor antagonist (G129R)- based fusion proteins were developed including G129R fusions with an angiogenesis inhibitor (endostatin), an immune system modulator (interleukin 2), and a modified truncated cytotoxin (PE38KDEL). Each fusion protein was designed to target the PRLR-positive cells via the G129R moiety and at the same time attack a hallmark common to cancer cells via the second moiety. In this study, we tested the efficacy of the three fusion proteins as a combination therapy in an aggressive but clinically relevant mouse tumor model. To test the feasibility and to optimize a treatment regimen, allografts of a mammary carcinoma cell line (McNeuA) derived from an MMTV-neu transgenic mouse were first used. Growth of the allografts was significantly retarded by regimens which combined all three fusion proteins. In addition, a significant increase in cytotoxic CD8+ T cells was observed within the tumors of the combination treated groups. After establishing the dosing regimen, two doses of cocktail treatment (low and high doses administered twice weekly) along with individual component controls were administered to female MMTV-neu transgenic mice after surgical removal of a naturally occurring tumor. The average tumor recurrence time was significantly delayed in both low and high combination treatment groups in comparison to the no treatment control group (34, 50 and 18 days, respectively). The total number of lung metastases was also significantly decreased in both combination treatment groups. In conclusion, using G129R-based fusion proteins to target mammary carcinomas and to tackle multiple hallmarks of cancer at the same time was an effective strategy for treating HER2-postive mammary cancer in this mouse tumor model.

Cell and Growth Factor-Loaded Keratin Hydrogels for Treatment of Volumetric Muscle Loss in a Mouse Model.

Wounds to the head, neck, and extremities have been estimated to account for ∼84% of reported combat injuries to military personnel. Volumetric muscle loss (VML), defined as skeletal muscle injuries in which tissue loss results in permanent functional impairment, is common among these injuries. The present standard of care entails the use of muscle flap transfers, which suffer from the need for additional surgery when using autografts or the risk of rejection when cadaveric grafts are used. Tissue engineering (TE) strategies for skeletal muscle repair have been investigated as a means to overcome current therapeutic limitations. In that regard, human hair-derived keratin (KN) biomaterials have been found to possess several favorable properties for use in TE applications and, as such, are a viable candidate for use in skeletal muscle repair. Herein, KN hydrogels with and without the addition of skeletal muscle progenitor cells (MPCs) and/or insulin-like growth factor 1 (IGF-1) and/or basic fibroblast growth factor (bFGF) were implanted in an established murine model of surgically induced VML injury to the latissimus dorsi (LD) muscle. Control treatments included surgery with no repair (NR) as well as implantation of bladder acellular matrix (BAM). In vitro muscle contraction force was evaluated at two months postsurgery through electrical stimulation of the explanted LD in an organ bath. Functional data indicated that implantation of KN+bFGF+IGF-1 (n = 8) enabled a greater recovery of contractile force than KN+bFGF (n = 8)***, KN+MPC (n = 8)**, KN+MPC+bFGF+IGF-1 (n = 8)**, BAM (n = 8)*, KN+IGF-1 (n = 8)*, KN+MPCs+bFGF (n = 9)*, or NR (n = 9)**, (*p < 0.05, **p < 0.01, ***p < 0.001). Consistent with the physiological findings, histological evaluation of retrieved tissue revealed much more extensive new muscle tissue formation in groups with greater functional recovery (e.g., KN+IGF-1+bFGF) when compared with observations in tissue from groups with lower functional recovery (i.e., BAM and NR). Taken together, these findings further indicate the general utility of KN biomaterials in TE and, moreover, specifically highlight their potential application in the treatment of VML injuries.

Keratin Hydrogel Enhances In Vivo Skeletal Muscle Function in a Rat Model of Volumetric Muscle Loss.

Volumetric muscle loss (VML) injuries exceed the considerable intrinsic regenerative capacity of skeletal muscle, resulting in permanent functional and cosmetic deficits. VML and VML-like injuries occur in military and civilian populations, due to trauma and surgery as well as due to a host of congenital and acquired diseases/syndromes. Current therapeutic options are limited, and new approaches are needed for a more complete functional regeneration of muscle. A potential solution is human hair-derived keratin (KN) biomaterials that may have significant potential for regenerative therapy. The goal of these studies was to evaluate the utility of keratin hydrogel formulations as a cell and/or growth factor delivery vehicle for functional muscle regeneration in a surgically created VML injury in the rat tibialis anterior (TA) muscle. VML injuries were treated with KN hydrogels in the absence and presence of skeletal muscle progenitor cells (MPCs), and/or insulin-like growth factor 1 (IGF-1), and/or basic fibroblast growth factor (bFGF). Controls included VML injuries with no repair (NR), and implantation of bladder acellular matrix (BAM, without cells). Initial studies conducted 8 weeks post-VML injury indicated that application of keratin hydrogels with growth factors (KN, KN+IGF-1, KN+bFGF, and KN+IGF-1+bFGF, n = 8 each) enabled a significantly greater functional recovery than NR (n = 7), BAM (n = 8), or the addition of MPCs to the keratin hydrogel (KN+MPC, KN+MPC+IGF-1, KN+MPC+bFGF, and KN+MPC+IGF-1+bFGF, n = 8 each) (p < 0.05). A second series of studies examined functional recovery for as many as 12 weeks post-VML injury after application of keratin hydrogels in the absence of cells. A significant time-dependent increase in functional recovery of the KN, KN+bFGF, and KN+IGF+bFGF groups was observed, relative to NR and BAM implantation, achieving as much as 90% of the maximum possible functional recovery. Histological findings from harvested tissue at 12 weeks post-VML injury documented significant increases in neo-muscle tissue formation in all keratin treatment groups as well as diminished fibrosis, in comparison to both BAM and NR. In conclusion, keratin hydrogel implantation promoted statistically significant and physiologically relevant improvements in functional outcomes post-VML injury to the rodent TA muscle.

Development and Characterization of a 3D Printed, Keratin-Based Hydrogel.

Keratin, a naturally-derived polymer derived from human hair, is physiologically biodegradable, provides adequate cell support, and can self-assemble or be crosslinked to form hydrogels. Nevertheless, it has had limited use in tissue engineering and has been mainly used as casted scaffolds for drug or growth factor delivery applications. Here, we present and assess a novel method for the printed, sequential production of 3D keratin scaffolds. Using a riboflavin-SPS-hydroquinone (initiator-catalyst-inhibitor) photosensitive solution we produced 3D keratin constructs via UV crosslinking in a lithography-based 3D printer. The hydrogels obtained have adequate printing resolution and result in compressive and dynamic mechanical properties, uptake and swelling capacities, cytotoxicity, and microstructural characteristics that are comparable or superior to those of casted keratin scaffolds previously reported. The novel keratin-based printing resin and printing methodology presented have the potential to impact future research by providing an avenue to rapidly and reproducibly manufacture patient-specific hydrogels for tissue engineering and regenerative medicine applications.

Keratin hydrogel carrier system for simultaneous delivery of exogenous growth factors and muscle progenitor cells.

Ideal material characteristics for tissue engineering or regenerative medicine approaches to volumetric muscle loss (VML) include the ability to deliver cells, growth factors, and molecules that support tissue formation from a system with a tunable degradation profile. Two different types of human hair-derived keratins were tested as options to fulfill these VML design requirements: (1) oxidatively extracted keratin (keratose) characterized by a lack of covalent crosslinking between cysteine residues, and (2) reductively extracted keratin (kerateine) characterized by disulfide crosslinks. Human skeletal muscle myoblasts cultured on coatings of both types of keratin had increased numbers of multinucleated cells compared to collagen or Matrigel(TM) and adhesion levels greater than collagen. Rheology showed elastic moduli from 10(2) to 10(5) Pa and viscous moduli from 10(1) to 10(4) Pa depending on gel concentration and keratin type. Kerateine and keratose showed differing rates of degradation due to the presence or absence of disulfide crosslinks, which likely contributed to observed differences in release profiles of several growth factors. In vivo testing in a subcutaneous mouse model showed that keratose hydrogels can be used to deliver mouse muscle progenitor cells and growth factors. Histological assessment showed minimal inflammatory responses and an increase in markers of muscle formation. © 2015 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 104B: 864-879, 2016.

The role of human prolactin and its antagonist, G129R, in mammary gland development and DMBA-initiated tumorigenesis in transgenic mice.

Human prolactin (hPRL) has been implicated to have a pathological role in breast cancer and play a critical role in mammary gland development. The hPRL antagonist, G129R, has been shown to induce breast cancer cell apoptosis. 9,10-Dimethyl-1,2-benzanthracene (DMBA), a potent mammary gland carcinogen, induces hormone responsive mammary tumor formation in rodents. To investigate the effects of hPRL and its counterpart, G129R, on mammary gland development and tumorigenesis, transgenic mice that express hPRL or G129R under the regulation of the metallothionein (Mt) promoter were generated. Mammary glands from virgin female transgenic mice at the ages of 12, 24, and 36 weeks were used to compare the effect of hPRL and G129R in various developmental stages. Mammary gland whole mount comparisons between transgenic mice and their littermates revealed a significant increase in ductal branching and lobular bud formation in hPRL transgenic mice; whereas a drastic decrease in ductal branching and lobular bud formation was observed in the mammary glands of G129R transgenic mice. In addition, total RNA isolated from the mammary glands of transgenic mice at the three different ages was analyzed on Affymetrix GeneChip Mouse Expression 430A chips (MOE430A). Microarray data revealed alteration to the gene expression levels, greatest at 12 and 36 weeks. Furthermore, hPRL and G129R transgenic mice, as well as their littermates, were treated with multiple doses of DMBA and the rate of mammary tumor formation and survival were compared. The tumor rates in the G129R transgenic mice were significantly reduced (18% at 28 weeks) as compared to that of either NTG (39%) or hPRL (40%). On the other hand, the tumor appearance is significantly earlier in the PRL transgenic group as compared to that of controls. Taken together, the data further confirmed the inhibitory effects of G129R in mammary gland development, which translates to a resistance to DMBA-initiated breast tumorigenesis.

Ciprofloxacin-Loaded Keratin Hydrogels Prevent Pseudomonas aeruginosa Infection and Support Healing in a Porcine Full-Thickness Excisional Wound.

Objective: Cutaneous wound infection can lead to impaired healing, multiple surgical procedures, and increased hospitalization time. We tested the effectiveness of keratin-based hydrogels (termed "keratose") loaded with ciprofloxacin to inhibit infection and support healing when topically administered to porcine excision wounds infected with Pseudomonas aeruginosa. Approach: Using a porcine excisional wound model, 10 mm full-thickness wounds were inoculated with 106 colony-forming units of P. aeruginosa and treated on days 1 and 3 postinoculation with ciprofloxacin-loaded keratose hydrogels. Bacteria enumeration and wound healing were assessed on days 3, 7, and 11 postinjury. Results: Ciprofloxacin-loaded keratose hydrogels reduced the amount of P. aeruginosa in the wound bed by 99.9% compared with untreated wounds on days 3, 7, and 11 postinjury. Ciprofloxacin-loaded keratose hydrogels displayed decreased wound contraction and reepithelialization at day 7 postinjury. By day 11, wounds treated with ciprofloxacin-keratose hydrogels contained collagen-rich granulation tissue and myofibroblasts. Wounds treated with ciprofloxacin-loaded keratose hydrogels exhibited a transient increase in macrophages in the wound bed at day 7 postinjury that subsided by day 11. Innovation: Current therapies for wound infection include systemic antibiotics, which could lead to antibiotic resistance, and topical antimicrobial treatments, which require multiple applications and can delay healing. Here, we show that ciprofloxacin-loaded keratose hydrogels inhibit cutaneous wound infection without interfering with key aspects of the healing process including granulation tissue deposition and remodeling. Conclusions: Ciprofloxacin-loaded keratose hydrogels have the potential to serve as a point-of-injury antibiotic therapy that prevents infection and supports healing following cutaneous injury.

Asphalt exposure enhances neuropeptide levels in sensory neurons projecting to the rat nasal epithelium.

Asphalt fumes have been reported to produce nasal irritation in road workers. Since inhaled irritants can increase substance P (SP) production in airway neurons, the effects of asphalt fumes on SP production in trigeminal ganglia (TG) sensory neurons innervating the nasal mucosa were investigated. The effects of asphalt fumes on nasal mucosal innervation were examined by measuring SP and calcitonin-gene-related peptide (CGRP) levels in rat TG neurons projecting to the nasal epithelium. Female Sprague-Dawley rats were exposed to asphalt fumes at 16.0 +/- 8.1mg /m3 for 5 consecutive days, 3.5 h/d. Inflammatory cells were measured in nasal cavity lavage fluid. SP and CGRP immunoreactivity (IR) was measured in the cell bodies of trigeminal ganglion sensory neurons projecting to the nasal cavity. A significant increase in neutrophils and macrophages was observed after asphalt fume exposure indicating an inflammatory response in the nasal cavity. The percentage of SP-IR neurons increased significantly in the asphalt-exposed rats, and the proportion of CGRP-IR neurons was also elevated following asphalt exposure. These results indicate that exposure to asphalt fumes produces inflammation and increases the levels of SP and CGRP in TG neurons projecting to the nasal epithelium. The findings are consistent with asphalt-induced activation of sensory C-fibers in the nasal cavity. Enhanced sensory neuropeptide release from nerve terminals in the nasal cavity may produce neurogenic inflammation associated with nasal irritation following exposure to asphalt fumes.

The humoral immune response of mice exposed to simulated road paving-like asphalt fumes.

Asphalt is a complex mixture of organic molecules, including polycyclic aromatic hydrocarbons (PAH), which have been reported to cause serious adverse health effects in humans. Workers in manufacturing and construction trades exposed to asphalt are potentially at risk for being exposed to asphalt fumes and PAHs. Epidemiological investigations have collected mounting evidence that chemicals found in asphalt fumes present carcinogenic and possibly immunotoxic hazards. Studies evaluating the immunotoxic effects of asphalt fume are limited due to the large number of variables associated with asphalt fume exposures. This work investigates the immuno-toxic effects of road paving-like asphalt fume by analyzing the in vivo IgM response to a T-dependent antigen after exposure to whole, vapor, and particulate phase road paving-like asphalt fumes and asphalt fume condensate. Systemic exposures via intraperitoneal injection of asphalt fume condensate (at 0.625 mg/kg) and the particulate phase (at 5 mg/kg) resulted in significant reductions in the specific spleen IgM response to SRBC. Pharyngeal aspiration of the asphalt fume condensate (at 5 mg/kg) also resulted in significant suppression of the IgM response to SRBC. A significant reduction in the specific spleen IgM activity was observed after inhalation exposure to whole asphalt fumes (35 mg/m(3)) and the vapor components (11 mg/m(3)). Dermal exposures to the asphalt fume condensate resulted in significant reductions in the total (at 50 mg/kg) and specific (at 250 mg/kg) spleen IgM response to SRBC. These results demonstrate that exposure to road paving-like asphalt fumes is immunosuppressive through systemic, respiratory, and dermal routes of exposure in a murine model and raise concerns regarding the potential for adverse immunological effects.

Additive effects of a prolactin receptor antagonist, G129R, and herceptin on inhibition of HER2-overexpressing breast cancer cells.

Breast cancers overexpressing human epidermal growth factor receptor 2 (HER2) have been reported to have higher proliferative and metastatic activity in the presence of autocrine prolactin (PRL), indicating potential cooperation between HER2 and the PRL receptor (PRLR) during breast cancer progression. PRL can induce the tyrosine phosphorylation of HER2 which stimulates mitogen-activated protein kinase (MAPK) activity. To determine if this transactivation of HER2 by PRL contributes to anti-HER2 therapy resistance we examined the potential of combining Herceptin with a PRLR antagonist, G129R, which inhibits PRL-induced signaling, as a novel therapeutic strategy. Two PRL-expressing human breast cancer cell lines (T-47D and BT-474) that overexpress PRLR and HER2 to different degrees were chosen for this study. The phosphorylation status of HER2 and activation of MAPK, signal transducers and activators of transcription (STAT), as well as phosphatidylinositol-3 kinase (PI3K) signaling cascades were examined in response to Herceptin, G129R or a combination of the two in either the absence or presence of exogenous PRL. As a single agent, Herceptin was more effective than G129R at inhibiting AKT phosphorylation; whereas, G129R was superior at blocking STAT3 and STAT5 activation. G129R was also able to directly inhibit the HER2 phosphorylation. The combination of Herceptin and G129R had an additive inhibitory effect on HER2 and MAPK phosphorylation, confirming that the MAPK signaling is a converging pathway shared by both HER2 and the PRLR. Combination of Herceptin and G129R also additively inhibited cell proliferation in vitro and in vivo as measured by inhibition of the growth of T-47D and BT-474 xenografts in athymic nude mice. We conclude that an anti-HER2 and anti-PRLR regimen may offer a new approach to treat HER2-overexpressing breast cancers.

Model predictions of the recruitment of lung units and the lung surface area-volume relationship during inflation.

Experimental evidence suggests that the lung behaves as if it is composed of a large population of units which are recruited and derecruited during lung expansion and contraction. This study combines two previous models in order to estimate the probability distribution function describing lung unit opening pressures and the resulting alveolar surface area-volume relationship of the excised rat lung during inflation. Results indicate that the opening pressures of lung units during inflation can be described by a normal distribution. The end-expiratory pressure (EEP) has a large effect on the number of lung units that open during inflation and the properties of the area-volume relationship of the lung, but the distribution of opening pressures of individual lung units is fairly consistent regardless of EEP. This study also presents evidence that when the normalized lung area-volume relationship is represented by the equation [A(L)]N = [phiV(L)]N(n) during inflation from the closed state, the expansion coefficient n is between 0.86 and 1. This result supports the theory that, for inflation from EEPs below 4 cmH2O, lung expansion occurs in part by the recruitment of lung units and not solely by the expansion of open units.