Apparatus and Method for Rapid Detection of Acoustic Anisotropy in Cartilage.

PURPOSE: Articular cartilage is known to be mechanically anisotropic. In this paper, the acoustic anisotropy of bovine articular cartilage and the effects of freeze-thaw cycling on acoustic anisotropy were investigated. METHODS: We developed apparatus and methods that use a magnetic L-shaped sample holder, which allowed minimal handling of a tissue, reduced the number of measurements compared to previous studies, and produced highly reproducible results. RESULTS: SOS was greater in the direction perpendicular to the articular surface compared to the direction parallel to the articular surface (N=17, P = 0.00001). Average SOS was 1,758 ± 107 m/s perpendicular to the surface, and 1,617 ± 55 m/s parallel to it. The average percentage difference in SOS between the perpendicular and parallel directions was 8.2% (95% CI: 5.4% to 11%). Freeze-thaw cycling did not have a significant effect on SOS (P>0.4). CONCLUSION: Acoustic measurement of tissue properties is particularly attractive for work in our laboratory since it has the potential for nondestructive characterization of the properties of developing engineered cartilage. Our approach allowed us to observe acoustic anisotropy of articular cartilage rapidly and reproducibly. This property was not significantly affected by freeze-thawing of the tissue samples, making cryopreservation practical for these assays.

In an in-vitro model using human fetal membranes, 17-α hydroxyprogesterone caproate is not an optimal progestogen for inhibition of fetal membrane weakening.

BACKGROUND: The progestogen 17-α hydroxyprogesterone caproate (17-OHPC) is 1 of only 2 agents recommended for clinical use in the prevention of spontaneous preterm delivery, and studies of its efficacy have been conflicting. We have developed an in-vitro model to study the fetal membrane weakening process that leads to rupture in preterm premature rupture of the fetal membranes (pPROM). Inflammation/infection associated with tumor necrosis factor-α (TNF-α) induction and decidual bleeding/abruption associated thrombin release are leading causes of preterm premature rupture of the fetal membranes. Both agents (TNF-α and thrombin) cause fetal membrane weakening in the model system. Furthermore, granulocyte-macrophage colony-stimulating factor (GM-CSF) is a critical intermediate for both TNF-α and thrombin-induced fetal membrane weakening. In a previous report, we demonstrated that 3 progestogens, progesterone, 17-alpha hydroxyprogesterone (17-OHP), and medroxyprogesterone acetate (MPA), each inhibit both TNF-α- and thrombin-induced fetal membrane weakening at 2 distinct points of the fetal membrane weakening pathway. Each block both the production of and the downstream action of the critical intermediate granulocyte-macrophage colony-stimulating factor. OBJECTIVE: The objective of the study was to characterize the inhibitory effects of 17-OHPC on TNF-α- and thrombin-induced fetal membrane weakening in vitro. STUDY DESIGN: Full-thickness human fetal membrane fragments from uncomplicated term repeat cesarean deliveries were mounted in 2.5 cm Transwell inserts and cultured with/without 17-alpha hydroxyprogesterone caproate (10-9 to 10-7 M). After 24 hours, medium (supernatant) was removed and replaced with/without the addition of tumor necrosis factor-alpha (20 ng/mL) or thrombin (10 U/mL) or granulocyte-macrophage colony-stimulating factor (200 ng/mL). After 48 hours of culture, medium from the maternal side compartment of the model was assayed for granulocyte-macrophage colony-stimulating factor and the fetal membrane fragments were rupture strength tested. RESULTS: Tumor necrosis factor-alpha and thrombin both weakened fetal membranes (43% and 62%, respectively) and increased granulocyte-macrophage colony-stimulating factor levels (3.7- and 5.9-fold, respectively). Pretreatment with 17-alpha hydroxyprogesterone caproate inhibited both tumor necrosis factor-alpha- and thrombin-induced fetal membrane weakening and concomitantly inhibited the induced increase in granulocyte-macrophage colony-stimulating factor in a concentration-dependent manner. However, contrary to our prior reports regarding progesterone and other progestogens, 17-alpha hydroxyprogesterone caproate did not also inhibit granulocyte-macrophage colony-stimulating factor-induced fetal membrane weakening. CONCLUSION: 17-Alpha hydroxyprogesterone caproate blocks tumor necrosis factor-alpha- and thrombin-induced fetal membrane weakening by inhibiting the production of granulocyte-macrophage colony-stimulating factor. However, 17-alpha hydroxyprogesterone caproate did not also inhibit granulocyte-macrophage colony-stimulating factor-induced weakening. We speculate that progestogens other than 17-alpha hydroxyprogesterone caproate may be more efficacious in preventing preterm premature rupture of the fetal membranes-related spontaneous preterm birth.

Progesterone inhibits in vitro fetal membrane weakening.

OBJECTIVE: Inflammation/infection and abruption are leading causes of preterm premature rupture of the membranes. Recently, we identified granulocyte-macrophage colony-stimulating factor (GM-CSF) as a critical mediator of both tumor necrosis factor-α- (TNF; modeling inflammation) and thrombin-induced (modeling abruption) weakening of the fetal membranes. We found that (1) TNF and thrombin both induced GM-CSF in the choriodecidua, (2) blockade of GM-CSF action with neutralizing antibodies inhibited both TNF- and thrombin-induced fetal membrane weakening, and (3) GM-CSF alone induced fetal membrane weakening. GM-CSF is thus part of an overlap of the inflammation and abruption-induced fetal membrane weakening pathways. The effects of progesterone analogs on the pathways by which fetal membranes are weakened have not been investigated. We examined the effects of progesterone, medroxyprogesterone acetate (MPA) and 17α-hydroxyprogesterone (HP) on TNF- and thrombin-induced fetal membrane weakening. STUDY DESIGN: Full-thickness fetal membranes from uncomplicated term repeat cesarean deliveries were mounted in Transwell inserts in Minimum Essential Medium alpha and incubated at 37°C in 5% CO2. The choriodecidua side of the fetal membrane fragments were preincubated with progesterone, MPA, HP, or vehicle for 24 hours. Fetal membranes were then exposed to TNF, thrombin, or GM-CSF on the choriodecidua side for an additional 48 hours. The fetal membrane tissues were then strength tested, and medium from the choriodecidua and amnion compartments was assayed for GM-CSF content. RESULTS: TNF and thrombin both weakened fetal membranes and elevated media GM-CSF levels on the choriodecidua side of the fetal membrane. Pretreatment with progesterone, MPA, or HP inhibited both TNF- and thrombin-induced fetal membrane weakening and also inhibited the induced increase in GM-CSF. GM-CSF decreased fetal membrane rupture strength by 68%, which was inhibited by progestogen pretreatment with a potency order: progesterone

Depth-dependent shear behavior of bovine articular cartilage: relationship to structure.

The mechanical behavior of bovine articular cartilage in shear was measured and related to its structure through the depth of the tissue. To make these measurements, we designed an apparatus that could apply controlled shear displacement and measure the resulting shear force on cartilage specimens. Shear displacement and shear strain were obtained from confocal images of photobleached lines on fluorescently stained deformed samples. Depth-dependent collagen structure was obtained using compensated polarized light microscopy. Depth-dependent shear behavior and structure of samples from two animals were measured (group A and B). Both animals were 18-24 months old, which is the range in which they are expected reach skeletal maturity. In mature samples (group A), the stiffest region was located beneath the superficial zone, and the most compliant region was found in the radial zone. In contrast, in samples that were in the process of maturing (group B) the most compliant region was located in the superficial zone. Compensated polarized light microscopy suggested that the animal from which the group A samples were obtained was skeletally mature, whereas the animal yielding the group B samples was in the process of maturing. Compensated polarized light microscopy was an important adjunct to the mechanical shear behavior in that it provided a means to reconcile differences in observed shear behavior in mature and immature cartilage. Although samples were harvested from two animals, there were clear differences in structure and shear mechanical behavior. Differences in the depth-dependent shear strain were consistent with previous studies on mature and immature samples and, based on the structural variation between mature and immature articular cartilage, their mechanical behavior differences can be tenable. These results suggest that age, as well as species and anatomic location, need to be considered when reporting mechanical behavior results.

Multimodal evaluation of tissue-engineered cartilage.

Tissue engineering (TE) has promise as a biological solution and a disease modifying treatment for arthritis. Although cartilage can be generated by TE, substantial inter- and intra-donor variability makes it impossible to guarantee optimal, reproducible results. TE cartilage must be able to perform the functions of native tissue, thus mechanical and biological properties approaching those of native cartilage are likely a pre-requisite for successful implantation. A quality-control assessment of these properties should be part of the implantation release criteria for TE cartilage. Release criteria should certify that selected tissue properties have reached certain target ranges, and should be predictive of the likelihood of success of an implant in vivo. Unfortunately, it is not currently known which properties are needed to establish release criteria, nor how close one has to be to the properties of native cartilage to achieve success. Achieving properties approaching those of native cartilage requires a clear understanding of the target properties and reproducible assessment methodology. Here, we review several main aspects of quality control as it applies to TE cartilage. This includes a look at known mechanical and biological properties of native cartilage, which should be the target in engineered tissues. We also present an overview of the state of the art of tissue assessment, focusing on native articular and TE cartilage. Finally, we review the arguments for developing and validating non-destructive testing methods for assessing TE products.

Mechanism of Human Fetal Membrane Biomechanical Weakening, Rupture and Potential Targets for Therapeutic Intervention.

Using a novel in vitro model system combining biochemical/histologic with bioengineering approaches has provided significant insights into the physiology of fetal membrane weakening and rupture along with potential mechanistic reasons for lack of efficacy of currently clinically used agents to prevent preterm premature rupture of the membranes (pPROM) and preterm births. Likewise, the model has also facilitated screening of agents with potential for preventing pPROM and preterm birth.

The nonlinear relationship between speed of sound and compression in articular cartilage: Measurements and modeling.

We measured speed of sound in bovine articular cartilage as a function of compressive strain. Using techniques we developed, it was possible to apply strain starting from the unstrained, full height of a sample. Our measurements showed that speed of sound was not a monotonic function of strain as reported in earlier investigations. Speed increased with increasing strain over a range of lower strains. It reached a maximum, and then decreased as the strain increased further. These results were corroborated using a model of wave propagation in deformable porous materials. Using this model, we also established conditions under which a maximum in the speed would exist for samples in compression. Our measurements and analysis resolve the conflicting results reported in previous studies.

Granulocyte macrophage colony stimulating factor (GM-CSF), the critical intermediate of inflammation-induced fetal membrane weakening, primarily exerts its weakening effect on the choriodecidua rather than the amnion.

INTRODUCTION: We have previously demonstrated two associations of PPROM, (1) inflammation/infection (modeled by tumor necrosis factor (TNF)) and (2) decidual bleeding (modeled by thrombin), both decrease fetal membrane (FM) rupture strength in-vitro. Furthermore, Granulocyte-Macrophage-Colony-Stimulating-Factor (GM-CSF) induced by both TNF and thrombin is a critical intermediate, necessary and sufficient for weakening by either agent. The amnion is the strength component of FM and must weaken for FM to rupture. It is unclear whether GM-CSF weakens amnion (AM) directly, or initially targets choriodecidua (CD) which secondarily releases agents to act on amnion. METHODS: Full thickness FM fragments were treated with/without GM-CSF. Some were preincubated with alpha-lipoic acid (LA), a known inhibitor of FM weakening. The FM fragments were then strength-tested. Separately, FM fragments were initially separated to AM and CD. AM fragments were cultured with Medium ± GM-CSF and then strength-tested. In other experiments, CD fragments were cultured with Medium, GM-CSF, LA, or LA + GM-CSF. Conditioned medium from each group was then incubated with AM. AM was then strength-tested. Matrix Metalloproteinases (MMPs) and Tissue Inhibitors of Matrix Metalloproteinases (TIMPs) were analyzed by Mutiplex Elisa. RESULTS: GM-CSF weakened intact FM which was blocked by LA. GM-CSF did not weaken isolated AM. However, GM-CSF conditioned CD media weakened AM and this weakening was inhibited by LA. GM-CSF treatment of CD increased MMPs 2, 9, and 10, and decreased TIMPs 1-3. LA reversed these effects. CONCLUSIONS: GM-CSF does not weaken amnion directly; GM-CSF acts on CD to increase proteases and decrease anti-proteases which secondarily weaken the amnion.

In an in-vitro model using human fetal membranes, α-lipoic acid inhibits inflammation induced fetal membrane weakening.

INTRODUCTION: We established an in-vitro model for the study of human fetal membrane (FM) weakening leading to pPROM. In this model, granulocyte-macrophage colony-stimulating factor (GM-CSF) is a critical intermediate for both tumor necrosis factor-α (TNF; modeling infection/inflammation) and thrombin (modeling decidual bleeding/abruption)-induced weakening. Thus, inhibitors of FM weakening can be categorized as targeting GM-CSF production, GM-CSF downstream action, or both. Most progestogens inhibit both, except 17-α hydroxyprogesterone caproate which inhibits FM weakening at only one point, GM-CSF production. α-lipoic acid (LA), an over-the-counter dietary supplement, has also been previously shown to inhibit TNF and thrombin induced FM weakening. OBJECTIVE: To determine the point of action of LA inhibition of FM weakening. METHODS: FM fragments were mounted in Transwell inserts and preincubated with/without LA/24 h, then with/without addition of TNF, thrombin or GM-CSF. After 48 h, medium was assayed for GM-CSF, and FM fragments were rupture-strength tested. RESULTS: TNF and thrombin both weakened FM and increased GM-CSF levels. GM-CSF also weakened FM. LA inhibited both TNF and thrombin induced FM weakening and concomitantly inhibited the increase in GM-CSF in a concentration-dependent manner. In addition, LA inhibited GM-CSF induced FM weakening in a concentration dependent manner. CONCLUSIONS: LA blocks TNF and thrombin induced FM weakening at two points, inhibiting both GM-CSF production and downstream action. Thus, we speculate that LA may be a potential standalone therapeutic agent, or supplement to current therapy for prevention of pPROM related spontaneous preterm birth, if preclinical studies to examine feasibility and safety during pregnancy are successfully accomplished.

Rapid Detection of Shear-Induced Damage in Tissue-Engineered Cartilage Using Ultrasound.

Previous investigations have shown that tissue-engineered articular cartilage can be damaged under a combination of compression and sliding shear. In these cases, damage was identified in histological sections after a test was completed. This approach is limited, in that it does not identify when damage occurred. This especially limits the utility of an assay for evaluating damage when comparing modifications to a tissue-engineering protocol. In this investigation, the feasibility of using ultrasound (US) to detect damage as it occurs was investigated. US signals were acquired before, during, and after sliding shear, as were stereomicroscope images of the cartilage surface. Histology was used as the standard for showing if a sample was damaged. We showed that US reflections from the surface of the cartilage were attenuated due to roughening following sliding shear. Furthermore, it was shown that by scanning the transducer across a sample, surface roughness and erosion following sliding shear could be identified. Internal delamination could be identified by the appearance of new echoes between those from the front and back of the sample. Thus, it is feasible to detect damage in engineered cartilage using US.

Thyroxine Increases Collagen Type II Expression and Accumulation in Scaffold-Free Tissue-Engineered Articular Cartilage.

Low collagen accumulation in the extracellular matrix is a pressing problem in cartilage tissue engineering, leading to a low collagen-to-glycosaminoglycan (GAG) ratio and poor mechanical properties in neocartilage. Soluble factors have been shown to increase collagen content, but may result in a more pronounced increase in GAG content. Thyroid hormones have been reported to stimulate collagen and GAG production, but reported outcomes, including which specific collagen types are affected, are variable throughout the literature. Here we investigated the ability of thyroxine (T4) to preferentially stimulate collagen production, as compared with GAG, in articular chondrocyte-derived scaffold-free engineered cartilage. Dose response curves for T4 in pellet cultures showed that 25 ng/mL T4 increased the total collagen content without increasing the GAG content, resulting in a statistically significant increase in the collagen-to-GAG ratio, a fold change of 2.3 ± 1.2, p < 0.05. In contrast, another growth factor, TGFß1, increased the GAG content in excess of threefold more than the increase in collagen. In large scaffold-free neocartilage, T4 also increased the total collagen/DNA at 1 month and at 2 months (fold increases of 2.1 ± 0.8, p < 0.01 and 2.1 ± 0.4, p < 0.001, respectively). Increases in GAG content were not statistically significant. The effect on collagen was largely specific to collagen type II, which showed a 2.8 ± 1.6-fold increase of COL2A1 mRNA expression (p < 0.01). Western blots confirmed a statistically significant increase in type II collagen protein at 1 month (fold increase of 2.2 ± 1.8); at 2 months, the fold increase of 3.7 ± 3.3 approached significance (p = 0.059). Collagen type X protein was less than the 0.1 µg limit of detection. T4 did not affect COL10A1 and COL1A2 gene expression in a statistically significant manner. Biglycan mRNA expression increased 2.6 ± 1.6-fold, p < 0.05. Results of this study show that an optimized dosage of T4 is able to increase collagen type II content, and do so preferential to GAG. Moreover, the upregulation of COL2A1 gene expression and type II collagen protein accumulation, without a concomitant increase in collagens type I or type X, signifies a direct enhancement of chondrogenesis of hyaline articular cartilage without the induction of terminal differentiation.

Cartilage tissue engineering for laryngotracheal reconstruction: comparison of chondrocytes from three anatomic locations in the rabbit.

Tissue engineering may provide a technique to generate cartilage grafts for laryngotracheal reconstruction in children. The present study used a rabbit model to characterize cartilage generated by a candidate tissue engineering approach to determine, under baseline conditions, which chondrocytes in the rabbit produce tissue-engineered cartilage suitable for in vivo testing in laryngotracheal reconstruction. We characterized tissue-engineered cartilage generated in perfused bioreactor chambers from three sources of rabbit chondrocytes: articular, auricular, and nasal cartilage. Biomechanical testing and histological, immunohistochemical, and biochemical assays were performed to determine equilibrium unconfined compression (Young's) modulus, and biochemical composition and structure. We found that cartilage samples generated from articular or nasal chondrocytes lacked the mechanical integrity and stiffness necessary for completion of the biomechanical testing, but five of six auricular samples completed the biomechanical testing (moduli of 210 +/- 93 kPa in two samples at 3 weeks and 100 +/- 65 kPa in three samples at 6 weeks). Auricular samples showed more consistent staining for proteoglycans and collagen II and had significantly higher glycosaminoglycan (GAG) content and concentration and higher collagen content than articular or nasal samples. In addition, the delayed gadolinium enhanced MRI of cartilage (dGEMRIC) method revealed variations in GAG spatial distribution in auricular samples that were not present in articular or nasal samples. The results indicate that, for the candidate tissue engineering approach under baseline conditions, only rabbit auricular chondrocytes produce tissue-engineered cartilage suitable for in vivo testing in laryngotracheal reconstruction. The results also suggest that this and similar tissue engineering approaches must be optimized for each potential source of chondrocytes.

The force required to rupture fetal membranes paradoxically increases with acute in vitro repeated stretching.

OBJECTIVE: The purpose of this study was to determine whether acute repetitive stretching causes fetal membranes (FM) weakening. STUDY DESIGN: Cesarean or vaginally delivered FM were repeatedly stretched and thereafter subjected to rupture testing. Rupture strength (RS), work to rupture (WR), and stiffness were determined. Unstretched FM were compared with stretched FM. RESULTS: In the cesarean group, FM stretched to 50% or 75% of the baseline (unstretched) RS for 10-20 cycles of 10 seconds each paradoxically showed increased RS and stiffness. WR decreased compared with baseline. Detailed analysis revealed that even a single stretch cycle initiated these changes to physical properties. Vaginally delivered FM showed similar changes in physical properties, as did separated amnion. CONCLUSION: Acute stretch forces do not directly cause FM weakening.

Scaffold-free cartilage subjected to frictional shear stress demonstrates damage by cracking and surface peeling.

Scaffold-free engineered cartilage is being explored as a treatment for osteoarthritis. In this study, frictional shear stress was applied to determine the friction and damage behaviour of scaffold-free engineered cartilage, and tissue composition was investigated as it related to damage. Scaffold-free engineered cartilage frictional shear stress was found to exhibit a time-varying response similar to that of native cartilage. However, damage occurred that was not seen in native cartilage, manifesting primarily as tearing through the central plane of the constructs. In engineered cartilage, cells occupied a significantly larger portion of the tissue in the central region where damage was most prominent (18 ± 3% of tissue was comprised of cells in the central region vs 5 ± 1% in the peripheral region; p < 0.0001). In native cartilage, cells comprised 1-4% of tissue for all regions. Average bulk cellularity of engineered cartilage was also greater (68 × 103 ± 4 × 103 vs 52 × 103 ± 22 × 103 cells/mg), although this difference was not significant. Bulk tissue comparisons showed significant differences between engineered and native cartilage in hydroxyproline content (8 ± 2 vs 45 ± 3 µg HYP/mg dry weight), solid content (12.5 ± 0.4% vs 17.9 ± 1.2%), shear modulus (0.06 ± 0.02 vs 0.15 ± 0.07 MPa) and aggregate modulus (0.12 ± 0.03 vs 0.32 ± 0.14 MPa), respectively. These data indicate that enhanced collagen content and more uniform extracellular matrix distribution are necessary to reduce damage susceptibility. Copyright © 2014 John Wiley & Sons, Ltd.

Micrometer scale guidance of mesenchymal stem cells to form structurally oriented large-scale tissue engineered cartilage.

Current clinical methods to treat articular cartilage lesions provide temporary relief of the symptoms but fail to permanently restore the damaged tissue. Tissue engineering, using mesenchymal stem cells (MSCs) combined with scaffolds and bioactive factors, is viewed as a promising method for repairing cartilage injuries. However, current tissue engineered constructs display inferior mechanical properties compared to native articular cartilage, which could be attributed to the lack of structural organization of the extracellular matrix (ECM) of these engineered constructs in comparison to the highly oriented structure of articular cartilage ECM. We previously showed that we can guide MSCs undergoing chondrogenesis to align using microscale guidance channels on the surface of a two-dimensional (2-D) collagen scaffold, which resulted in the deposition of aligned ECM within the channels and enhanced mechanical properties of the constructs. In this study, we developed a technique to roll 2-D collagen scaffolds containing MSCs within guidance channels in order to produce a large-scale, three-dimensional (3-D) tissue engineered cartilage constructs with enhanced mechanical properties compared to current constructs. After rolling the MSC-scaffold constructs into a 3-D cylindrical structure, the constructs were cultured for 21days under chondrogenic culture conditions. The microstructure architecture and mechanical properties of the constructs were evaluated using imaging and compressive testing. Histology and immunohistochemistry of the constructs showed extensive glycosaminoglycan (GAG) and collagen type II deposition. Second harmonic generation imaging and Picrosirius red staining indicated alignment of neo-collagen fibers within the guidance channels of the constructs. Mechanical testing indicated that constructs containing the guidance channels displayed enhanced compressive properties compared to control constructs without these channels. In conclusion, using a novel roll-up method, we have developed large scale MSC based tissue-engineered cartilage that shows microscale structural organization and enhanced compressive properties compared to current tissue engineered constructs. STATEMENT OF SIGNIFICANCE: Tissue engineered cartilage constructs made with human mesenchymal stem cells (hMSCs), scaffolds and bioactive factors are a promising solution to treat cartilage defects. A major disadvantage of these constructs is their inferior mechanical properties compared to the native tissue, which is likely due to the lack of structural organization of the extracellular matrix of the engineered constructs. In this study, we developed three-dimensional (3-D) cartilage constructs from rectangular scaffold sheets containing hMSCs in micro-guidance channels and characterized their mechanical properties and metabolic requirements. The work led to a novel roll-up method to embed 2-D microscale structures in 3-D constructs. Further, micro-guidance channels incorporated within the 3-D cartilage constructs led to the production of aligned cell-produced matrix and enhanced mechanical function.

Separation of amnion from choriodecidua is an integral event to the rupture of normal term fetal membranes and constitutes a significant component of the work required.

OBJECTIVE: This study was undertaken to determine the sequence of events that occur during fetal membrane (FM) rupture and to compare the biophysical properties of intact FM with its separated individual components (amnion and choriodecidua). STUDY DESIGN: FM physical properties were determined with computerized, specially adapted, industrial, strength testing equipment and the rupture sequence (in vitro) video documented. Separated individual FM component properties were compared with those of reapproximated components, and of intact FMs. RESULTS: The sequence of FM rupture was (1) FM components stretch together under load; (2) amnion separates from choriodecidua; (3) choriodecidua ruptures; (4) amnion distends further, nonelastically; and (5) amnion ruptures. In all FMs tested, amnion was stronger, stiffer, and more ductile than choriodecidua. The sum of work required to rupture separated FM components (amnion + choriodecidua), or reapproximated components, was significantly less than that of intact FMs. CONCLUSION: Separation of amnion from choriodecidua occurs as part of normal term FM rupture. FMs become significantly weaker as a result of this separation.

Nondestructive Techniques to Evaluate the Characteristics and Development of Engineered Cartilage.

In this review, methods for evaluating the properties of tissue engineered (TE) cartilage are described. Many of these have been developed for evaluating properties of native and osteoarthritic articular cartilage. However, with the increasing interest in engineering cartilage, specialized methods are needed for nondestructive evaluation of tissue while it is developing and after it is implanted. Such methods are needed, in part, due to the large inter- and intra-donor variability in the performance of the cellular component of the tissue, which remains a barrier to delivering reliable TE cartilage for implantation. Using conventional destructive tests, such variability makes it near-impossible to predict the timing and outcome of the tissue engineering process at the level of a specific piece of engineered tissue and also makes it difficult to assess the impact of changing tissue engineering regimens. While it is clear that the true test of engineered cartilage is its performance after it is implanted, correlation of pre and post implantation properties determined non-destructively in vitro and/or in vivo with performance should lead to predictive methods to improve quality-control and to minimize the chances of implanting inferior tissue.

Validation of Ultrasound Elastography Imaging for Nondestructive Characterization of Stiffer Biomaterials.

Ultrasound elastography (UE) has been widely used as a "digital palpation" tool to characterize tissue mechanical properties in the clinic. UE benefits from the capability of noninvasively generating 2-D elasticity encoded maps. This spatial distribution of elasticity can be especially useful in the in vivo assessment of tissue engineering scaffolds and implantable drug delivery platforms. However, the detection limitations have not been fully characterized and thus its true potential has not been completely discovered. Characterization studies have focused primarily on the range of moduli corresponding to soft tissues, 20-600 kPa. However, polymeric biomaterials used in biomedical applications such as tissue scaffolds, stents, and implantable drug delivery devices can be much stiffer. In order to explore UE's potential to assess mechanical properties of biomaterials in a broader range of applications, this work investigated the detection limit of UE strain imaging beyond soft tissue range. To determine the detection limit, measurements using standard mechanical testing and UE on the same polydimethylsiloxane samples were compared and statistically evaluated. The broadest detection range found based on the current optimized setup is between 47 kPa and 4 MPa which exceeds the modulus of normal soft tissue suggesting the possibility of using this technique for stiffer materials' mechanical characterization. The detectable difference was found to be as low as 157 kPa depending on sample stiffness and experimental setup.

The physiology of fetal membrane weakening and rupture: Insights gained from the determination of physical properties revisited.

Rupture of the fetal membranes (FM) is precipitated by stretch forces acting upon biochemically mediated, pre-weakened tissue. Term FM develop a para-cervical weak zone, characterized by collagen remodeling and apoptosis, within which FM rupture is thought to initiate. Preterm FM also have a weak region but are stronger overall than term FM. Inflammation/infection and decidual bleeding/abruption are strongly associated with preterm premature FM rupture (pPROM), but the specific mechanisms causing FM weakening-rupture in pPROM are unknown. There are no animal models for study of FM weakening and rupture. Over a decade ago we developed equipment and methodology to test human FM strength and incorporated it into a FM explant system to create an in-vitro human FM weakening model system. Within this model TNF (modeling inflammation) and Thrombin (modeling bleeding) both weaken human FM with concomitant up regulation of MMP9 and cellular apoptosis, mimicking the characteristics of the spontaneous FM rupture site. The model has been enhanced so that test agents can be applied directionally to the choriodecidual side of the FM explant consistent with the in-vivo situation. With this enhanced system we have demonstrated that the pathways involving inflammation/TNF and bleeding/Thrombin induced FM weakening overlap. Furthermore GM-CSF production was demonstrated to be a critical common intermediate step in both the TNF and the Thrombin induced FM weakening pathways. This model system has also been used to test potential inhibitors of FM weakening and therefore pPROM. The dietary supplement α-lipoic acid and progestogens (P4, MPA and 17α-hydroxyprogesterone) have been shown to inhibit both TNF and Thrombin induced FM weakening. The progestogens act at multiple points by inhibiting both GM-CSF production and GM-CSF action. The use of a combined biomechanical/biochemical in-vitro human FM weakening model system has allowed the pathways of fetal membrane weakening to be delineated, and agents that may be of clinical use in inhibiting these pathways to be tested.

Determination of poroelastic properties of cartilage using constrained optimization coupled with finite element analysis.

The feasibility of determining biphasic material properties using a finite element model of stress relaxation coupled with two types of constrained optimization to match measured data was investigated. Comparison of these two approaches, a zero-order method and a gradient-based algorithm, validated the predicted material properties. Optimizations were started from multiple different initial guesses of material properties (design variables) to establish the robustness of the optimization. Overall, the optimal values are close to those found by Cohen et al. (1998) but these small differences produced a marked improvement in the fit to the measured stress relaxation. Despite the greater deviation in the optimized values obtained from the zero-order method, both optimization procedures produced material properties that gave equally good overall fits to the measured data. Furthermore, optimized values were all within the expected range of material properties. Modeling stress relaxation using the optimized material properties showed an excellent fit to the entire time history of the measured data.