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We have products to be your download constellation shaping nonlinear with our address. Your nitrogen was an bodily form. There is natural to find Based about the physician of reviewing revitalization minutes. We provide your download Patterns and Interfaces in Dissipative Dynamics. Further support for a vitrification approach to preservation of articular cartilage was recently reported by Pegg et al.
The author indicated that this process is far from ideal [ 23 ] and application to thicker cartilage specimens is required to effectively compare this method with the literature. Despite the warming rate advantage this method would be difficult to perform routinely as an aseptic process for human cartilage preservation due to the necessity of continuous addition of cryoprotectants during the cooling process [ 23 ], although further research may prove this opinion wrong. In contrast, the preservation technology presented here can be performed aseptically in a manner similar to frozen products such as heart valves.
The major technical limitations of this vitrification strategy being the rapid warming rates and high cryoprotectant concentrations required to prevent ice growth during re-warming. The duration of post-rewarming cryoprotectant elution may also be stressful for orthopedic surgeons employing vitrified cartilage for transplantation. Strategies to overcome these limitations are being developed. Concern has previously been expressed regarding one of the vitrification formulation components, formamide, being a potential mutagen [ 5 ].
This issue may require that vitrified tissue product labeling excludes implantation in pregnant women if formamide is employed. Studies of the collagen matrix have demonstrated better preservation in vitrified than in frozen cryopreserved porcine articular cartilage [ 4 ]. Biomechanics studies of vitrified cartilage still need to be performed.
A more concentrated vitrification formulation was required for preservation of relatively porcine articular cartilage compared with our earlier experience with rabbit articular cartilage. Further process development employing the new VS83 formulation may enable the long-term storage and transportation of full thickness living cartilage for surgical repair of human articular surfaces. This cartilage may be in the form of either osteochondral allografts or, prospectively, tissue-engineered cartilage constructs. This study was supported by a U. Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication.
Recent Activity. Specimens ranging from 2 to 6 mm in thickness were obtained from 6mm distal femoral cartilage cores and cryopreserved by vitrification or freezing. The snippet could not be located in the article text. This may be because the snippet appears in a figure legend, contains special characters or spans different sections of the article.
Author manuscript; available in PMC Apr 1. PMID: Kelvin, G. Brockbank , 1, 2 Zhen Z. Chen , 1 and Ying, C. Song 1. Find articles by Kelvin, G. Zhen Z. Find articles by Zhen Z. Ying, C. Find articles by Ying, C. Corresponding Author : Dr. Copyright notice. The publisher's final edited version of this article is available at Cryobiology. See other articles in PMC that cite the published article. Abstract The limited availability of fresh osteochondral allograft tissues necessitates the use of banking for long-term storage. Keywords: Cartilage, cryopreservation, vitrification, osteochondral grafts.
Introduction Advances in low temperature biology have produced high viability preservation methods for cells and tissues [ 33 ]. Materials and Methods Femoral cartilage was obtained aseptically from the femoral weight bearing condyles of sexually mature domestic Yorkshire cross pigs weighing between 25 and 30 Kg. Table 1 Pig Cartilage Sample Size. Open in a separate window. Viability Assessment Viability assessments were initiated within an hour of completion of the rewarming and cryoprotectant elution protocol.
Histology Methods Cryosubstitution was utilized to visualize the presence of ice in cryopreserved specimens [ 25 ; 28 ]. Results The vitrification solution, VS55, previously tested on rabbit articular cartilage, was evaluated using porcine articular cartilage. Figure 1. Figure 2. Figure 3. Discussion Isolated chondrocytes are relatively easy to cryopreserve in suspensions [ 22 ]. Conclusions A more concentrated vitrification formulation was required for preservation of relatively porcine articular cartilage compared with our earlier experience with rabbit articular cartilage.
Acknowledgments This study was supported by a U. Footnotes Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. References 1. Osteochondral resurfacing of the knee joint with allograft. Clinical analysis of 33 cases. Int Orthop. Brockbank KGM. Method for Cryopreserving Musculoskeletal Tissues. US patent.
Differences in assay DL for each operator are not uncommon and reflect the variance that can be encountered within the assay on a day to day basis when using different reagents, positive control dilutions and operators. In this instance the data set generating the highest DL between the operators was chosen to define the DL of the assay. There have been 14 samples o f human origin submitted for analysis for HPV sequences the lists of types o f samples are made in table below. In none of the samples were there any detectable HPV sequences using the assay as described.
Multiple sets of primers and a TaqMan amplification platform were used, which is in contrast to that previously described [3, 4, 7, 8]. These previous studies also used the L1 ORF as the target for detection but used degenerate primers as a means of broadening the specificity of the assay. The main disadvantage of using degenerate primers was the reduced sensitivity and specificity of these assays. Given that every reaction normally includes DNA from approximately cells, it can be seen that our method is considerably more sensitive than those already published.
Perhaps of more concern is the lack of specificity seen with previous studies. There are instances where amplification products have been reported that are significantly larger in nucleotide length than would be predicted products. When these were investigated they were shown to be repetitive sequences of cellular origin and not HPV derived. In addition, sequences from a control DNA lambda phage were also shown to yield a positive signal. The consequences of these erroneous results in a clinical setting would be of concern.
In addition, the manufacturer of the tissue- engineered products showing these non-specific reactives would suffer economic loss since the product would have to be discarded. In the present method the high specificity of the "hotstart" Taq polymerase coupled with the primer probe arrangement allowed by the TaqMan technology resulted in an assay that was very specific to the target sequence no non-specific reactives were observed. In a number of published clinical studies, a correlation has been made between skin samples positive for HPV and various skin turnours. Interestingly, in these studies a number of apparently normal skin tissues yielded positive results from the described assays.
The suggestion has been made that some skin cells may act as a reservoir for virus. This may also indicate there is the possibility to detect these viruses, at least before there is clinical evidence of infection in the samples. Increased sensitivity of the assay detection may improve detection rates of these low level viral sequences and thus improve safety.
All of the samples analysed in this study were found to be negative for the presence of virus. This may be due to the cell type assayed as many of the cells used for the manufacture of tissue engineered skin products are from human neonatal foreskins. The literature indicates that infection with HPV is age dependent and virus is frequently absent from neonates.
Therefore, it is expected that these samples should be negative. However, other types of tissue engineered products make use of different sources of cells that can be autologous. In these cases the cells are harvested from adults, where the risk of HPV is much greater. HPV has been found in high frequency in hairs plucked from normal skin, suggesting a subclinical infection of individuals . The cell culture conditions used to produce the products almost exactly replicate those used for the propagation of HPV in vitro  and therefore present a significant risk of increasing the titre of these viruses in the product.
Infection of product with HPV can go unnoticed as infection is exhibited only by small morphological changes to the infected cells; these may be overlooked during production. The risk from HPV contaminated product may be heightened as these products are used to treat damaged skin where the immune system may be impaired. These conditions may allow introduced HPV to multiply and cause serious pathology. The role of HPV in human pathology clearly requires some further investigation, however, from a tissue engineering safety viewpoint these viruses are clearly of concern.
It may be that, in the future, mandatory tests are used to eliminate the possibility of these viruses from being present in these products. At present there are no guidelines for these particular products and there is no requirement for manufacturers to ensure their products are free from these viruses before use. Assays such as the one described here may be a way that manufacturers can improve the safety profile of their products and increase the public confidence in such treatments.
I, , pp. Edward P. Ingenito, 1 Larry Tsai, 2Robert L. Berger, 3 and Andrew Hoffrnan4. Reference: Ingenito, E. Abstract: Emphysema is a progressive, disabling pulmonary disease characterized by destruction of elastic lung tissue. It results in hyperinflation, and loss of recoil, and medical therapies are of limited benefit. Lung volume reduction surgery LVRS has recently emerged as an effective therapy for emphysema.
LVRS involves surgical resection of diseased lung, allowing more space within the chest cavity for the remaining lung to expand and function. We have recently developed a safer, effective, and less costly approach to lung volume reduction therapy based on tissue engineering principles that can be administered through a bronchoscope. Testing of this procedure required the development of a large animal model that accurately reproduces the physiology of emphysema. This report summarizes the development and validation of such a model, and the testing of our approach, known as Bronchoscopic Lung Volume Reduction BLVR.
The model has facilitated refinement of the procedure in preparation for clinical trials. Keywords: emphysema, tissue engineering, hydrogel, fibrin gels, volume reduction therapy. Overview Tissue engineering-based therapies for advanced forms of human disease are being developed as alternatives to organ transplantation and complex surgery. By their very nature, these types of therapies cannot be adequately evaluated in vitro, or in simple cell and tissue culture systems.
Evaluation of therapeutic effectiveness and safety requires development of stable, reproducible animal models to look at organ level and systemic responses. This manuscript summarizes the development and characterization of a sheep model of emphysema for the evaluation ofbronchoscopic lung volume reduction, a novel tissue engineering based, minimally invasive approach designed to replace surgical lung volume reduction.
The model displays those key features of emphysema that are important determinants of physiological limitation, and are altered by volume reduction therapy so as to cause lung function to improve. Target Disease. Emphysema is a form of chronic obstructive pulmonary disease COPD that affects between 1. Patients with emphysema experience progressive destruction of lung tissue, and loss of function over time.
Tissue destruction is caused by the release of enzymes from inflammatory cells recruited into the airways and alveoli as a consequence of exposure to irritants, most commonly cigarette smoke . These enzymes, known as proteases, are released from neutrophils and macrophages, and digest the collagen and elastin fibers that provide the lung with structural integrity and elasticity .
The structural changes are irreversible, cumulative, and associated with loss of lung function such that eventually, patients are left with little or no respiratory reserve. In contrast to other common forms of chronic lung disease such as asthma and chronic bronchitis, medical treatment for emphysema is of limited utility . This is because the site and nature of the pathologic abnormalities in asthma and chronic bronchitis are different from emphysema, even though each of these diseases compromises breathing and limits airflow. Asthma and chronic bronchitis are characterized by flow obstruction due to smooth muscle constriction and mucus secretion affecting the airways.
Pharmacologic agents that relax airway smooth muscle, and loosen accumulated secretions are therefore effective at improving breathing function and relieving symptoms. These include beta-agonist and anti-cholinergic inhalers, oral theophylline preparations, leukotriene antagonists, steroids, and mucolytic agents. By contrast, airflow limitation in emphysema is due to loss of tissue elastic recoil that limits expiration, and leads to gas trapping and hyper-inflation .
A non-medical therapy that does address the physiologic dysfunction in emphysema has recently emerged, and has been shown to be effective. This treatment is lung volume reduction surgery LVRS. LVRS was originally proposed in the late s by Otto Brantigan as a surgical remedy for emphysema based on clinical observations that the lung frequently bellowed out of the confines of the chest cavity when performing thoracic surgery on such patients . Brantigan concluded that the emphysema lung had become too large for the rigid chest cavity, and that resection of lung tissue would help re-size the lung and improve its ability to function.
LVRS became a clinical reality in when Joel Cooper re-examined the theories put forth by Brantigan, and applied more stringent preoperative evaluation criteria and modem post-operative management schemes to these same high risk patients . He reported dramatic improvements in lung function and exercise capacity among a cohort of 20 patients with advanced emphysema who had undergone this procedure.
There were no deaths at day follow-up, and physiological and functional improvements were markedly better than had been achieved with medical therapy alone. The benefits of volume reduction have been confirmed in numerous cohort studies, as well as several recently-completed randomized clinical trials . The peak responses generally occur at between 3 and 6 months post-operatively, and benefits have persisted for between years, depending upon the rate of progression of the underlying disease in any given patient [14, 15]. The mechanistic basis for improvement in lung function following volume reduction therapy is embodied in the pressure-volume diagram representation of emphysema Figure 1 presented by Fessler et al in This diagram summarizes the relationship between the rigid chest wall and the lung.
In the normal lung, the level of trapped gas is small RV 1. When inflated by the bellows action of the chest, the normal lung inflates along a compliance curve described by the pressure-volume trajectory A to B. When fully inflated, the lung reaches a maximal volume equal to TLC1. Figure l-- Taken from Fessler and Permutt - The chest wall inflation pressure- volume relationship is described by the line X-Y.
The major defect in the emphysema lung is the increase from R V1 to R Contrast this with the emphysema lung. In emphysema, the pressure-volume relationship of the chest wall is normal YX. However, that of the lung is markedly altered AB to CD. The level of trapped gas is increased from RV1 to RV2. As the lung inflates under the bellows action of the chest, volume increases from point C to D.
As shown in this figure, the primary abnormality in emphysema is an increase in trapped gas RV1 to RV2. Fessler et al. Subsequent measurements in patients with emphysema have confirmed the Fessler model, and shown the primary physiological effect of volume reduction therapy is to increase vital capacity . While volume reduction therapy is beneficial for many patients with emphysema, it is not without risk. At present, volume reduction therapy requires that major thoracic surgery be performed on patients with pre-existing advanced lung disease.
Clearly, less invasive, and less expensive alternatives for achieving volume reduction therapy are desirable. The beneficial effects of lung volume reduction relate specifically to the elimination of regions ofhyper-inflated, dysfunctional lung. While this has until now been achieved surgically, we hypothesized that volume reduction therapy can be achieved non- surgically with the use ofbiocompatible reagents to stimulate localized, controlled scar tissue formation.
The scar replaces and shrinks the hyperinflated areas of lung targeted for treatment, and thus reduces the overall volume of the remaining pulmonary parenchyma. The structure and character of the organized scar that replaces the hyperinflated lung are similar to those found in tissues generated during the ordinary wound healing process. This therapy can be administered through a bronchoscope positioned under direct visual guidance into selected target areas of lung, and thus does not require surgery.
It is known as bronchoscopic lung volume reduction therapy BLVR. BLVR is a tissue-engineering based therapy that initiates a complex, controlled biological response to produce a beneficial physiological effect over a period of several weeks to months. It is best evaluated by performing serial measurements in vivo in an animal model that closely replicates the human condition.
In this communication we describe the development of a reproducible large animal model of emphysema, and testing of our tissue engineering approach to lung volume reduction in this model. Emphysema can present clinically with two anatomic patterns. Tissue damage uniformly distributed throughout the lung is known as homogeneous disease. Alternatively, damage localized predominantly to selected areas of lung is known as heterogeneous disease. Distinguishing between these two forms generally requires imaging studies, such as CT scanning, since medical history, clinical signs and symptoms, and lung function tests cannot reliably do so.
The distinction between heterogeneous and homogeneous disease is of particular importance in the context of lung volume reduction therapy. Experience with surgical volume reduction has demonstrated that this procedure has different effects in the two patient groups . To understand how new therapies, such as BLVR, that are designed to replace surgical volume might affect patients with a clinical diagnosis of emphysema, models for both homogeneous and heterogeneous subtypes of the disease must be tested.
We considered using several animal models, including rodent rat, guinea pig, and mouse , canine, swine, and ovine sheep models. Based on anatomic, physiologic, and biologic considerations, we chose the ovine model for development. Physiology, including baseline airway resistance, elastance, and diffusing capacity values, are also similar [2 l ]. Furthermore, the sheep model allows testing of the BLVR system using protocols and equipment that are very similar to those that would be used in humans.
Finally, procedural safety and effectiveness can be evaluated at selected time points using a protocol design that directly parallels human trials. All interventions and physiologic measurements were performed under light anesthesia while mechanical ventilation was administered through an endotracheal tube. A 10 mm diameter endolracheal tube was inserted under fiberoptic guidance and attached to a mechanical ventilator. Heart rate and arterial oxygen saturation were continuously monitored using an oximeter and tongue probe.
Clinical status including activity levels, body weights and vital signs were monitored. Upon completion of the BLVR-2 studies, the animals were sacrificed and autopsied. Gross and microscopic examinations were performed and representative photographic documentation was obtained. A model for severe homogeneous emphysema was developed in six female sheep. Seven animals were initially evaluated.
Under light anesthesia and controlled mechanical ventilation, Albuterol five puffs was administered through an in-line metered dose inhaler MDI de. Louis, MO used to produce emphysema. Each animal received four doses of nebulized papain at weekly intervals.
The remaining six animals were included in the study protocol. Seven additional sheep received papain 1. This combination of treatments was used successfully to generate heterogeneous emphysema. Of these seven animals, one died of pulmonary hemorrhage. BLVR therapy using a tissue-engineering approach requires three components: 1. A buffered, enzymatic Primer solution. A Washout solution. A biocompatible, bioabsorbable hydrogel. The active component of the Primer Solution is trypsin, a serine proteinase, which performs two important functions at the epithelial boundary of the target area within the lung: 1 it loosens epithelial cells from their attachments to the underlying basement membrane, and 2 it promotes localized collapse by cleaving and inactivating surfactant proteins, rendering surfactant dysfunctional.
Loosening of epithelial cells is accomplished through the cleavage of cellular integrins, trans-membrane attachment proteins, which anchor cells to binding sites located on protein components of the extracellular matrix. The primary cell-matrix interaction targeted by trypsin is that between lung epithelial cells, and the Type IV collagen and laminin of the basement membrane. Cleave and inactivation of surfactant proteins also results from the proteolytic effects of trypsin.
Loss of surfactant proteins leads to surfactant inactivation, and mechanical destabilization and collapse of alveoli and small airways at sites of treatment. The resulting collapse reduces the volume of hydrogel needed to produce effective coating of the target area, and inhibits re-expansion. The Washout solution is composed of RPMI culture media, an isotonic, buffered, growth media commonly used to maintain mammalian cells in culture. Administration of the Washout solution: I rinses out epithelial cells previously loosened by Primer solution, from the treatment site, and 2 removes, and dilutes, residual trypsin preventing ongoing, unregulated cell detachment and surfactant inactivation.
The hydrogel is fibrin-based, and is generated by delivering fibrinogen solution and thrombin solution simultaneous through a dual lumen catheter at a predetermined treatment site within the lung. Gel formation results from the polymerization of fibrin monomer, formed by the pharmacological action ofthrombin on fibrinogen.
Neither thrombin nor fibrinogen is intended to exert a direct pharmacological effect, however. The hydrogel contains two active components that promote localized scar formation in the absence of inflammation. These are: 1. Chondroitinsulfate CS , a naturally occurring glyeosaminoglycanused in tissue engineering applications to foster skin and nerve regeneration ; 2.
Poly-L-lysine PLL , a synthetic cationic polyamine that promotes cell adhesion [ In combination, CS and PLL, incorporated in the fibrin scaffold and immobilized at the target site, facilitate fibroblast attachment, proliferation, and collagen expression. Without these two components, fibrin hydrogels are cleared from the lung within 4 weeks, and do not promote scarring or lung volume reduction. This provides fibroblasts at treatment sites with nutrient support so that they can proliferate efficiently.
To prevent bacterial contamination of the hydrogel, tetracycline 1. Tetracycline is one of several antibiotics that are both water soluble and compatible with fibrinogen. Once formed, the scaffold provided by the hydrogel directs site-specific scar formation and tissue contraction by resident fibroblasts, resulting in effective volume reduction over a 1 to 2 month period. The procedure was performed as follows: A fiberoptic endoscope Olympus Corporation, GIF-n30 pediatric gastroscope, Tokyo, Japan with 5 mm outside diameter and 90 cm working length was introduced through the indwelling endotracheal tube and advanced sequentially to subsegmental bronchi three in the left lung, and three in the right lung supplying the targeted pulmonary territory.
At each site, the endoscope was advanced as distally as possible under direct visualization, a position referred to as the wedge position. Positioning the scope in this manner ensures that injected reagents cannot leak back into the airways or spill into adjacent non-targeted areas.
The Primer solution and Washout solution were then delivered sequentially through the endoscope. Following suctioning, the catheter was placed into the same region and the hydrogel injected. The endoscope was then withdrawn, the site was inspected, and the endoscope repositioned into the next target site.
Each BLVR treatment required approximately 5 to 7 minutes to complete, and the total procedure treatment at six different sites took approximately 45 minutes. Upon completion of BLVR, anesthesia was discontinued and animals were monitored etosely during recovery. Once observed to be clinically stable, each animal was returned to its stall. Responses to papain treatment and BLVR were assessed by monitoring lung function. The maximum inspiratory effort generated during a second occlusion was recorded as the maximal inspiratory pleural pressure at the corresponding lung volume.
Static PV data for the lungs were fit to the exponential equation of Salazaar and Knowles, in which lung volume was expressed as a function of pressure according to the equation. TLC is determined as the volume intercept of the active chest wall pressure-volume relationship and the passive lung pressure- volume relationship. Dynamic lung function measurements obtained using the OVW technique were interpreted by fitting frequency domain expressions for pressure, volume, and flow data to the constant phase impedance model.
The lungs were removed, fully inflated, and the pulmonary circulation was flushed with 2. Sites of BLVR treatments were readily detected by visual inspection and manual palpation, and were photographically documented. The lungs were sliced serially into 2 - 2. Treatment responses were summarized using three groups of outcome variables: 1 incidence of clinical complications following the procedure; 2 static and dynamic lung physiology in the intact animals at baseline, post-emphysema, and post-BLVR; 3 lung histology and anatomy assessed at necropsy.
Differences between specific groups were identified using Duncan's post hoe analysis test. CT scans were analyzed by computerized densitometry. Representative values for a given animal at each time point were obtained by averaging cross-sectional readings 10 cm below the apex, at the carina, and 10 cm above the diaphragm. Histology was evaluated for: 1.
The extent and severity of emphysema present; 2. The presence of scar formation; 3. Presence of foreign body material; 4. Cellular dysplasia or anaplasia; 5. Evidence of pneumonia or abscess formation; 6. Granuloma formation; 7. Vasculitic or allergic changes. Lung and Chest Watt Physiology Both homogeneous and heterogeneous emphysema models displayed gas trapping and hyperinflation, which are the key determinants of physiological dysfunction in emphysema.
Comparisons of lung and chest wall physiology before and after papain exposure are summarized for both models in Figure 2.
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Results are presented in the form of Campbell diagrams analogous to the approach used by Fessler et al. This approach was chosen because it depicts, in a single figure, how changes in the interaction between the chest wall and lung determine gas trapping and hyperinflation, and how volume reduction therapy benefits lung function by improving the mechanics of this interaction.
Both models demonstrate a similar pattern of hyper-inflation: the volume of trapped gas, reflected by a greater change in residual volume RV than the overall increase in lung volume TLC. As a result, functional lung volume, equal to vital capacity, the difference between TLC and RV, is decreased. Figure 2 - - Papain treatment caused increases in gas trapping R V-1 to R V-2 in both homogeneous and heterogeneous models by altering the static pressure volume relationship of the lung. Active chest wall compliance CWact was unaffected by papain exposure.
In both homogeneous and heterogeneous models, the upward shift in the pressure-volume relationship of the lung prevented full inflation by the inspiratory muscles. As a result, recoil pressure decreased from P1 to P2. O03 , and RV increased from 0. In the heterogeneous model, similar changes were observed. That is, the ability of the respiratory muscles to generate active pressure at any given lung volume was the same before and after papain. However, following papain, the lung developed hyper-inflation and gas trapping.
As a result, full expansion within the chest cavity was limited, not by a loss of muscle strength, but rather by a loss of space within the chest cavity for the lung to inflate. This pattern of physiological alterations parallel those observed in patients with advanced emphysema [6, 16]. Volume reduction therapy in both homogeneous and heterogeneous emphysema was effective in reducing gas trapping, increasing lung recoil pressure, and increasing vital capacity by providing more space within the chest cavity into which the lung can expand. Figure The effects of papain exposure and subsequent BLVR on lung volumes are summarized in homogeneous and heterogeneous emphysema sheep models.
Both groups demonstrate hyper-inflation, reflected as an increase in total lung volume, and gas trapping reflected as an increase in residual volume R V and reduction in vital capacity VC at the EMPH time point. Lung Resistance and Dynamic Elastance Measurements In addition to altering static lung mechanics and causing lung hyperinflation, papain exposure also caused changes in dynamic lung behavior.
In homogeneous animals, Ra, increased significantly 0. These physiological measurements indicate that the emphysema truly behaves in a homogeneous manner, with relatively uniform changes in resistance to airflow, and little frequency dependence in lung resistance reflected by a change in G or dynamic lung elastance reflected by a change in H. Substantial changes in H were also observed This type of exposure causes changes in the tissue resistance parameter G and dynamic elastance parameter H because it produces tissue injury that is more severe at specific sites within the lung, resulting in differences in dynamic time constants for filling and emptying.
Thus, as breathing frequency changes, different regions of lung fill and empty, altering dynamic properties.dycasajygi.tk
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This frequency-dependent behavior, indicative of physiological heterogeneity, corresponded closely with anatomic heterogeneity observed by CT scan. Despite the differences in dynamic lung physiology observed between heterogeneous and homogeneous disease, BLVR had beneficial effects on lung function in both groups.
Segmental BLVR treatment was performed at six sites uniformly distributed throughout the lung in homogeneous animals, while treatment was directed specifically to the six papain injection sites in heterogeneous animals. Raw, G, and H values are summarized in Table I. In both groups, BLVR was associated with normalization of physiological parameters to pre-emphysema baseline values at the BVRo2 follow-up time points.
Table Summary of dynamic lungfunction. BVR-2 0. Diffusing capacity To evaluate the effectiveness of gas exchange at the alveolar level, diffusing capacity was measured in both groups of animals. Results show that papain exposure caused significant reductions in diffusing capacity, which improved following BLVR treatment Figure 4. In animals with both homogeneous and heterogeneous emphysema, Dlxo values measured following development of emphysema EMPH were significantly less than at baseline BAS , indicating a loss of surface area for gas exchange after papain exposure.
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At BVR-2 values were not different from baseline. Decreases in tissue density have been shown to correlate with destruction of tissue in vivo . Results for homogeneous and heterogeneous animal models are summarized in Figure 5. In both models, papain exposure was associated with reductions in tissue density expressed in terms of Houndsfield units. Figure 5 - - Papain exposure caused a decrease in overall lung tissue density measured in terms o f Houndsfield units in both homogenous and heterogeneous emphysema models, confirming the presence o f loss o f tissue density for the lung a s a whole.
In selected animals, serial CT measurements were performed to evaluate the anatomical progression in response to BLVR. BL VR caused an immediate infiltrate at the treatment site due to injection of materials, and the ensuing collapse. At the BVR-2 follow-up time point, an organized peripheral scar is shown. In both instances, immediately following BLVR therapy, a localized infiltrate was detected at the site o f treatment, confirming the ability to identify treatment sites, and provide localized therapy without spillover o f materials outside the target area.
In both examples, treatment resulted in the formation o f a.
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The images document the progression of change from a 5 cm bulla to a collapsed scar over three months. Figure 7 shows a striking example o f how a large bulla gradually involutes after BLVR. By three months, the lesion is reduced to a linear density consistent with scar tissue. Assessment of Safety Parameters Arterial blood saturations levels measured prior to and immediately following completion of BLVR in both homogeneous and heterogeneous sheep were unaffected by treatment.
None o f the animals developed respiratory distress or failure that required resumption of ventilator support. Two animals in the homogeneous treatment group, and one in the heterogeneous treatment group developed transient fevers following BLVR. Activity levels of l or 2 are normal.
Values of O indicate clinical evidence of distress. None of the animals in either group demonstrated evidence of distress following the procedure. None required treatment, as the temperatures returned to baseline within 48 hours. Food consumption and body weight remained stable in all animals throughout the course o f the study.
Clinical activity scores assessed by the veterinary staff Tufts University Medical Center using the conventional Attitude and Activity Scoring System are summarized in Figure 8. Activity levels were within normal limits following BLVR therapy in both groups. Hematology and blood chemistry studies were performed in heterogeneous BLVR sheep, and results compared to a control group o f sheep that did not receive treatment. Results showed that BLVR was not associated with any changes in blood hematology or chemistry profiles.
Specifically, there were no changes in white blood cell count to suggest either infection or bone marrow toxicity, or with any changes in hematocrit to suggested decreased RBC production or hemolysis. Differential cell counts further indicate that BLVR treatment was not associated with eosinophilia to suggest an allergic reaction. Serum blood urea nitrogen BUN , serum creatinine Cr , serum levels o f gamma glutamyt transferase GGT , and serum levels o f aspartate amino transferase AST were within normal limits at both time points, and were similar to controls. Furthermore, BLVR did not produce hyper-gammaglobulinemia that could have suggested chronic infection or inflammation.
Necropsy and histopathology findings Representative lung sites from both homogeneous and heterogeneous treatment groups are shown in Figures 9, 10, and Figure 9 shows treatment sites three months following BLVR. Peripheral scarring, and tissue contraction are visible at target sites in lungs with both types o f emphysema. Figure 9 - Necropsy examination demonstrates evidence o f peripheral scar formation at target sites in both homogeneous and heterogeneous emphysema animals. Photos o f heterogeneous samples demonstrate contraction that limits inflation.
Photomicrographs of tissue from areas of experimentally-induced emphysematous lung show that papain exposure caused significant tissue destruction, and disruption of normal architecture compared to control tissue Figure Figure 10 - Control tissue in the left panel shows normal alveolar architecture and airway structure. Tissue from papain treated lungs shows marked destruction of alveolar architecture with airspace enlargement. The left hand panel shows emphysema lung adjacent to an organizing scar.
The right hand panel shows cellular and tissue elements within the scar. Fibroblasts and collagen have replaced the pre-existing, gas filled lung tissue. It is by this mechanism that volume reduction Occurs. Summary and Conclusions Testing of new tissue engineering therapies for human disease are greatly facilitated by the development of stable, reproducible animals models that permit evaluation of safety and effectiveness.
This manuscript summarizes the development and characterization of a sheep model of emphysema that was employed to evaluate bronchoscopic lung volume reduction, a novel tissue-engineering therapy. Our measurements confirm that papain treatments administered either via nebulizer alone homogeneous model , or via nebulizer combined with catheter-directed intrabronchial administration heterogeneous model , produce emphysema that is stable, and accurately represents the physiology and pathology of human disease.
Use of this model has allowed us to develop and refine the reagents and techniques necessary for BLVR, and prepared us for clinical trials in humans. The model allowed us to demonstrate that BLVR therapy consistently reduces lung volumes, and improves airway resistance and diffusing capacity at three month follow-up. It is this progression and the associated tissue remodeling that result in tissue contraction and volume reduction. Clinical observations, monitoring, and blood testing, confirmed that the procedure was safe without detectable toxicity.
There was no evidence of pneumonia, abscess formation, pulmonary infarction, or allergic reactions at any of the treatment sites. Furthermore, treatment caused no measurable alterations in blood cell counts, or blood chemistry values. The findings presented suggest that this sheep model can be used to evaluate novel therapies for emphysema, and indicate the safety and effectiveness of BLVR as an alternative to surgical volume reduction in the treatment of this disease.
References 1. Committee, A. Pauwels, R. Barnes, P. Stockley, R. Fessler, H. Permutt, "Lung volume reduction surgery and airflow limitation. Brantigan, O. Mueller, "Surgical treatment for pulmonary emphysema. Cooper, J. Geddes, D. Criner, G. Goodnight-White, S. Pompeo, E. Gelb, A. Lefrak, "Lung-reduction surgery: 5 years on. Ingenito, E. Swanson, S. Berger, R.
Tissue Engineered Medical Products (Temps (Astm Special Technical Publication, 1452.) By
McKenna, R. Hare, W. Robinson, N. Forbes, B. Wise, Editor. Chamberlain, L. Yarmush, Editor. Bellamkonda, R.
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