A chondroitinase-ABC and TGF-β1 treatment regimen for enhancing the mechanical properties of tissue-engineered fibrocartilage

The development of functionally equivalent fibrocartilage remains elusive despite efforts to engineer tissues such as knee meniscus, intervertebral disc and temporomandibular joint disc. Attempts to engineer these structures often fail to create tissues with mechanical properties on a par with native tissue, resulting in constructs unsuitable for clinical applications. The objective of this study was to engineer a spectrum of biomimetic fibrocartilages representative of the distinct functional properties found in native tissues. Using the self-assembly process, different co-cultures of meniscus cells and articular chondrocytes were seeded into agarose wells and treated with the catabolic agent chondroitinase-ABC (C-ABC) and the anabolic agent transforming growth factor-β1 (TGF-β1) via a two-factor (cell ratio and bioactive treatment), full factorial study design. Application of both C-ABC and TGF-β1 resulted in a beneficial or positive increase in the collagen content of treated constructs compared to controls. Significant increases in both the collagen density and fiber diameter were also seen with this treatment, increasing these values by 32 and 15%, respectively, over control values. Mechanical testing found the combined bioactive treatment to synergistically increase the Young’s modulus and ultimate tensile strength of the engineered fibrocartilages compared to controls, with values reaching the lower spectrum of those found in native tissues. Together, these data demonstrate that C-ABC and TGF-β1 interact to develop a denser collagen matrix better able to withstand tensile loading. This study highlights a way to optimize the tensile properties of engineered fibrocartilage using a biochemical and a biophysical agent together to create distinct fibrocartilages with functional properties mimicking those of native tissue.     

Maturational growth of self-assembled, functional menisci as a result of TGF-β1 and enzymatic chondroitinase-ABC stimulation

Replacement of the knee meniscus requires a material possessing adequate geometrical and biomechanical properties. Meniscal tissue engineering attempts have been unable to produce tissue with collagen content and biomechanical properties, particularly tensile properties, mimicking native menisci. In an effort to obtain the geometric properties and the maturational growth necessary for the recapitulation of biochemical and, thus, biomechanical properties, a scaffoldless cell-based system, the self-assembly process, was used in conjunction with the catabolic enzyme chondroitinase-ABC and TGF-β1. We show that combinations of these agents resulted in maturational growth as evidenced by synergistic enhancement of the radial tensile modulus by 5-fold and the compressive relaxation modulus by 68%, and additive increases of the compressive instantaneous modulus by 136% and Col/WW by 196%. This study shows that tissue engineering can produce a biomaterial that is on par with the biochemical and biomechanical properties of native menisci.     

TGF-β3-induced chondrogenesis in co-cultures of chondrocytes and mesenchymal stem cells on biodegradable scaffolds

In this work, it was hypothesized that co-cultures of articular chondrocytes (ACs) and mesenchymal stem cells (MSCs) would exhibit enhanced sensitivity to chondrogenic stimuli, such as TGF-β3, and would require a reduced concentration of TGF-β3 to achieve an equivalent level of chondrogenesis compared to monocultures of each cell type. Furthermore, it was hypothesized that compared to monocultures, the chondrogenic phenotype of AC/MSC co-cultures would be more stable upon the removal of TGF-β3 from the culture medium. These hypotheses were investigated by culturing ACs and MSCs alone and in a 1:3 ratio on electrospun poly(?-caprolactone) scaffolds. All cell populations were cultured for two weeks with 0, 1, 3, or 10 ng/ml of TGF-β3. After two weeks growth factor supplementation was removed, and the constructs were cultured for two additional weeks. Cell proliferation, extracellular matrix production, and chondrogenic gene expression were evaluated after two and four weeks. The results demonstrated that co-cultures of ACs and MSCs require a reduced concentration and duration of TGF-β3 exposure to achieve an equivalent level of chondrogenesis compared to AC or MSC monocultures. Thus, the present work implicates that the promise of co-cultures for cartilage engineering is enhanced by their robust phenotype and heightened sensitivity to TGF-β3.     

Mechano growth factor (MGF) and transforming growth factor (TGF)-β3 functionalized silk scaffolds enhance articular hyaline cartilage regeneration in rabbit model

Damaged cartilage has poor self-healing ability and usually progresses to scar or fibrocartilaginous tissue, and finally degenerates to osteoarthritis (OA). Here we demonstrated that one of alternative isoforms of IGF-1, mechano growth factor (MGF) acted synergistically with transforming growth factor β3 (TGF-β3) embedded in silk fibroin scaffolds to induce chemotactic homing and chondrogenic differentiation of mesenchymal stem cells (MSCs). Combination of MGF and TGF-β3 significantly increased cell recruitment up to 1.8 times and 2 times higher than TGF-β3 did in vitro and in vivo. Moreover, MGF increased Collagen II and aggrecan secretion of TGF-β3 induced hMSCs chondrogenesis, but decreased Collagen I in vitro. Silk fibroin (SF) scaffolds have been widely used for tissue engineering, and we showed that methanol treated pured SF scaffolds were porous, similar to compressive module of native cartilage, slow degradation rate and excellent drug released curves. At 7days after subcutaneous implantation, TGF-β3 and MGF functionalized silk fibroin scaffolds (STM) recruited more CD29+/CD44 + cells (P < 0.05). Similarly, more cartilage-like extracellular matrix and less fibrillar collagen were detected in STM scaffolds than that in TGF-β3 modified scaffolds (ST) at 2 months after subcutaneous implantation. When implanted into articular joints in a rabbit osteochondral defect model, STM scaffolds showed the best integration into host tissues, similar architecture and collagen organization to native hyaline cartilage, as evidenced by immunostaining of aggrecan, collagen II and collagen I, as well as Safranin O and Masson's trichrome staining, and histological evalution based on the modified O'Driscoll histological scoring system (P < 0.05), indicating that MGF and TGF-β3 might be a better candidate for cartilage regeneration. This study demonstrated that TGF-β3 and MGF functionalized silk fibroin scaffolds enhanced endogenous stem cell recruitment and facilitated in situ articular cartilage regeneration, thus providing a novel strategy for cartilage repair.     

The effect of epidermal growth factor on the incremental Young's moduli in the rat small intestine

Biomechanical remodelling of the rat small intestine after treatment with epidermal growth factor (EGF) subcutaneously for 2 days (n=6), 4 days (n=6), 7 days (n=6), and 14 days (n=4) was studied. The incremental circumferential, longitudinal and cross moduli close to the in vivo state were computed from bi-axial test data (combined inflation and axial stretching) by a least square method. The moduli in the circumferential direction and the longitudinal direction differed in all groups, i.e. the mechanical properties were anisotropic in both normal and EGF-treated rats. Time-dependent variation existed for the Young's moduli in all directions during EGF treatment (P<0.05). The circumferential modulus decreased during the first 7 days of EGF treatment and it almost remodelled back to that of the control group after 14 days treatment. The incremental modulus in the circumferential direction ranged between 17.4 and 24.2 kPa. The modulus in the longitudinal direction ranged between 22.9 and 32.4 kPa. The longitudinal modulus after 4 days EGF treatment was significantly larger than that of control group (P<0.02). The cross modulus decreased during the first 4 days of EGF treatment thereafter it increased to a maximum at 7 days. The values for the cross moduli were between 4.7 and 6.6 kPa. In conclusion, the mechanical properties in the intestinal wall are anisotropic and remodel during treatment with EGF.     

Intraspecies and Interspecies Comparison of the Compressive Properties of the Medial Meniscus

Quantification of the compressive material properties of the meniscus is of paramount importance, creating a "gold-standard" reference for future research. The purpose of this study was to determine compressive properties in six animal models (baboon, bovine, canine, human, lapine, and porcine) at six topographical locations. It was hypothesized that topographical variation of the compressive properties would be found in each animal model and that interspecies variations would also be exhibited. To test these hypotheses, creep and recovery indentation experiments were performed on the meniscus using a creep indentation apparatus and analyzed via a finite element optimization method to determine the material properties. Results show significant intraspecies and interspecies variation in the compressive properties among the six topographical locations, with the moduli exhibiting the highest values in the anterior portion. For example, the anterior location of the human meniscus has an aggregate modulus of 160 +/- 40 kPa, whereas the central and posterior portions exhibit aggregate moduli of 100 +/- 30 kPa. Interspecies comparison of the aggregate moduli identifies the lapine anterior location having the highest value (450 +/- 120 kPa) and the human posterior location having the lowest (100 +/- 30 kPa). These baseline values of compressive properties will be of help in future meniscal repair efforts.     

Growth factor priming of synovium-derived stem cells for cartilage tissue engineering

This study investigated the potential use of synovium-derived stem cells (SDSCs) as a cell source for cartilage tissue engineering. Harvested SDSCs from juvenile bovine synovium were expanded in culture in the presence (primed) or absence (unprimed) of growth factors (1?ng/mL transforming growth factor-β(1), 10?ng/mL platelet-derived growth factor-ββ, and 5?ng/mL basic fibroblast growth factor-2) and subsequently seeded into clinically relevant agarose hydrogel scaffolds. Constructs seeded with growth factor-primed SDSCs that received an additional transient application of transforming growth factor-β(3) for the first 21 days (release) exhibited significantly better mechanical and biochemical properties compared to constructs that received sustained growth factor stimulation over the entire culture period (continuous). In particular, the release group exhibited a Young's modulus (267±96?kPa) approaching native immature bovine cartilage levels, with corresponding glycosaminoglycan content (5.19±1.45%ww) similar to native values, within 7 weeks of culture. These findings suggest that SDSCs may serve as a cell source for cartilage tissue engineering applications.     

Transforming growth factor-beta 3 stimulates cartilage matrix elaboration by human marrow-derived stromal cells encapsulated in photocrosslinked carboxymethylcellulose hydrogels: potential for nucleus pulposus replacement

Degeneration of the nucleus pulposus (NP) has been implicated as a major cause of low back pain. Tissue engineering strategies using marrow-derived stromal cells (MSCs) have been used to develop cartilaginous tissue constructs, which may serve as viable NP replacements. Supplementation with growth factors, such as transforming growth factor-beta 3 (TGF-β3), has been shown to enhance the differentiation of MSCs and promote functional tissue development of such constructs. A potential candidate material that may be useful as a scaffold for NP tissue engineering is carboxymethylcellulose (CMC), a biocompatible, cost-effective derivative of cellulose. Photocrosslinked CMC hydrogels have been shown to support NP cell viability and promote phenotypic matrix deposition capable of maintaining mechanical properties when cultured in serum-free, chemically defined medium (CDM) supplemented with TGF-β3. However, MSCs have not been characterized using this hydrogel system. In this study, human MSCs (hMSCs) were encapsulated in photocrosslinked CMC hydrogels and cultured in CDM with and without TGF-β3 to determine the effect of the growth factor on the differentiation of hMSCs toward an NP-like phenotype. Constructs were evaluated for matrix elaboration and functional properties consistent with native NP tissue. CDM supplemented with TGF-β3 resulted in significantly higher glycosaminoglycan content (762.69±220.79 ng/mg wet weight) and type II collagen (COL II) content (6.25±1.64 ng/mg wet weight) at day 21 compared with untreated samples. Immunohistochemical analyses revealed uniform, pericellular, and interterritorial staining for chondroitin sulfate proteoglycan and COL II in growth factor-supplemented constructs compared with faint, strictly pericellular staining in untreated constructs at 21 days. Consistent with matrix deposition, mechanical properties of hydrogels treated with TGF-β3 increased over time and exhibited the highest peak stress in stress-relaxation (σ(pk)=1.489±0.389 kPa) at day 21 among all groups. Taken together, these results demonstrate that hMSCs encapsulated in photocrosslinked CMC hydrogels supplemented with TGF-β3 are capable of elaborating functional extracellular matrix consistent with the NP phenotype. Such MSC-laden hydrogels may have application in NP replacement therapies.     

Long-term intermittent compressive stimulation improves the composition and mechanical properties of tissue-engineered cartilage

Tissue engineering of articular cartilage is a promising alternative for cartilage repair. However, it has been difficult to develop tissue in vitro that mimicks native cartilage. Cartilaginous tissue formed in vitro does not accumulate enough extracellular matrix, is deficient in collagen, and possesses only a fraction of the mechanical properties of native cartilage. In this study, we investigated whether long-term intermittent compressive stimulation would improve the quality of the generated tissue. Chondrocyte cultures were established on the surface of porous calcium polyphosphate substrates and allowed to form cartilaginous tissue. In vitro-formed tissues were subjected to different stimulation protocols for 1 week. The optimal mechanical stimulation parameters identified in this short-term study were then applied to the cultures for up to 4 weeks. Mechanical stimulation applied at a 5% compressive amplitude at a frequency of 1 Hz for 400 cycles every second day resulted in the greatest increase in collagen synthesis (37 +/- 9% over control) while not significantly affecting proteoglycan synthesis (2 +/- 8% over control). This condition, applied to the chondrocyte cultures for 4 weeks, resulted in a significant increase in the amount of tissue that formed (stimulated, 2.4 +/- 0.2 mg dry wt; unstimulated, 1.61 +/- 0.08 mg dry wt). Stimulated tissues contained approximately 40% more collagen (stimulated, 590 +/- 58 microg; unstimulated, 420 +/- 42 microg), and 30% more proteoglycans (stimulated, 393 +/- 34 microg; unstimulated, 302 +/- 32 microg) as well as displaying a 2- to 3-fold increase in compressive mechanical properties (maximal equilibrium stress: stimulated, 10 +/- 1 kPa; unstimulated, 5 +/- 1 kPa; maximal equilibrium modulus: stimulated, 80 +/- 23 kPa; unstimulated, 24 +/- 6 kPa). The results of this study demonstrate that intermittent mechanical stimulation can increase collagen synthesis and, when applied over a 4-week period, can accelerate extracellular matrix accumulation as well as improve the material properties of the developed tissue. Interestingly, only short periods of mechanical stimulation (6 min every second day) were needed to affect the quality of cartilaginous tissue formed in vitro.     

The controlled presentation of TGF-β1 to hepatocytes in a 3D-microfluidic cell culture system

3D-microfluidic cell culture systems (3D-μFCCSs) support hepatocyte functions in vitro which can be further enhanced by controlled presentation of 100–200 pg/ml TGF-β1, thus mimicking the roles of supporting cells in co-cultures. Controlled presentation of TGF-β1 is achieved by either direct perfusion or in situ controlled release from gelatin microspheres immobilized in the 3D-μFCCS. Primary hepatocytes cultured for 7 days with the in situ controlled released TGF-β1 exhibited up to four-fold higher albumin secretion and two-fold higher phase I/II enzymatic activities, significantly improving the sensitivity of hepatocytes to acetaminophen-mediated hepatotoxicity, compared to hepatocytes cultured with directly perfused TGF-β1 or without TGF-β1. The controlled presentation of TGF-β1 enhanced hepatocyte functions in microfluidic systems without the complications of co-cultures, allowing for simplifications in drug testing and other hepatocyte-based applications.     

Evaluation of bone regeneration in implants composed of hollow HA microspheres loaded with transforming growth factor β1 in a rat calvarial defect model

Implants that serve simultaneously as an osteoconductive matrix and as a device for local growth factor delivery may be required for optimal bone regeneration in some applications. In the present study, hollow hydroxyapatite (HA) microspheres (106–150 μm) in the form of three-dimensional (3-D) scaffolds or individual (loose) microspheres were created using a glass conversion process. The capacity of the implants, with or without transforming growth factor β1 (TGF-β1), to regenerate bone in a rat calvarial defect model was compared. The 3-D scaffolds supported the proliferation and alkaline phosphatase activity of osteogenic MLO-A5 cells in vitro, showing their cytocompatibility. Release of TGF-β1 from the 3-D scaffolds into phosphate-buffered saline ceased after 2–3 days when ～30% of the growth factor was released. Bone regeneration in the 3-D scaffolds and the individual microspheres increased with time from 6 to 12 weeks, but it was significantly higher (23%) in the individual microspheres than in the 3-D scaffolds (15%) after 12 weeks. Loading with TGF-β1 (5 μg per defect) enhanced bone regeneration in the 3-D scaffolds and individual microspheres after 6 weeks, but had little effect after 12 weeks. 3-D scaffolds and individual microspheres with larger HA diameter (150–250 μm) showed better ability to regenerate bone. Based on these results, implants composed of hollow HA microspheres show promising potential as an osteoconductive matrix for local growth factor delivery in bone regeneration.     

Elasticity and viscoelasticity of embolization microspheres

The present study investigates the mechanical properties of three embolization microspheres (E-ms): tris-acryl gelatin microspheres (TG-ms), acrylamido polyvinyl alcohol microspheres (APVA-ms), and polyphosphazene-coated polymethylmethacrylate microspheres (PP-PMMA-ms). Compression and relaxation tests were performed on monolayers of particles and their Young's moduli and relaxation half times (RHTs) were determined. The elasticity of E-ms was evaluated by applying Hertz theory with the assumptions of incompressibility and a Poisson's ratio of 0.5. The Young's moduli of TG-ms, APVA-ms, and PP-PMMA-ms were 39.6±5.05 kPa, 18.8±4.00 kPa, and 13.6±1.98 kPa, respectively. The RHTs of TG-ms, APVA-ms, and PP-PMMA-ms were 52.3±5.56 s, 59.1±8.16 s, and 31.0±7.01 s, respectively. TG-ms have a high rigidity and deform slightly under a sustained compression since they have a high elasticity. PP-PMMA-ms are soft and deform a lot under sustained compression. They are more viscous than the other two microspheres. APVA-ms have intermediate material properties, having the same low rigidity as PP-PMMA-ms and being more elastic than TG-ms.     

Effects of auricular chondrocyte expansion on neocartilage formation in photocrosslinked hyaluronic acid networks

The overall objective of this study was to examine the effects of in vitro expansion on neocartilage formation by auricular chondrocytes photoencapsulated in a hyaluronic acid (HA) hydrogel as a next step toward the clinical application of tissue engineering therapies for treatment of damaged cartilage. Swine auricular chondrocytes were encapsulated either directly after isolation (p = 0), or after further in vitro expansion ( p = 1 and p = 2) in a 2 wt%, 50-kDa HA hydrogel and implanted subcutaneously in the dorsum of nude mice. After 12 weeks, constructs were explanted for mechanical testing and biochemical and immunohistochemical analysis and compared to controls of HA gels alone and native cartilage. The compressive equilibrium moduli of the p = 0 and p = 1 constructs (51.2 +/- 8.0 and 72.5 +/- 35.2 kPa, respectively) were greater than the p = 2 constructs (26.8 +/- 14.9 kPa) and the control HA gel alone (12.3 +/- 1.3 kPa) and comparable to auricular cartilage (35.1 +/- 12.2 kPa). Biochemical analysis showed a general decrease in glycosaminoglycan (GAG), collagen, and elastin content with chondrocyte passage, though no significant differences were found between the p = 0 and p = 1 constructs for any of the analyses. Histological staining showed intense and uniform staining for aggrecan, as well as greater type II collagen versus type I collagen staining in all constructs. Overall, this study illustrates that constructs with the p = 0 and p = 1 auricular chondrocytes produced neocartilage tissue that resembled native auricular cartilage after 12 weeks in vivo. However, these results indicate that further expansion of the chondrocytes (p = 2) can lead to compromised tissue properties.     

转化生长因子β与骨骼肌损伤修复的研究进展

肌肉的损伤修复是一个动态协调而又极其复杂的过程,TGF-β是一种内源性的生长因子,在肌肉损伤后通过重新编码肌肉细胞基因,抑制生肌细胞基因的表达,骨骼肌内TGF-β产生于损伤反应中,在损伤愈合的各个阶段均起作用并受TGF-β浓度的影响,TGF-β的生物学效应也受到其它生长因子的拮抗或协同作用,使用TGF-β阻断剂可以拮抗TGF-β进而减少肌肉纤维化,改善肌肉愈合质量。本文回顾了骨骼肌损伤修复与转化生长因子β的作用,并对转化生长因子β在肌肉损伤修复方面的应用前景进行了分析和探讨。     

Homologous structure-function relationships between native fibrocartilage and tissue engineered from MSC-seeded nanofibrous scaffolds

Understanding the interplay of composition, organization and mechanical function in load-bearing tissues is a prerequisite in the successful engineering of tissues to replace diseased ones. Mesenchymal stem cells (MSCs) seeded on electrospun scaffolds have been successfully used to generate organized tissues that mimic fibrocartilages such as the knee meniscus and the annulus fibrosus of the intervertebral disc. While matrix deposition has been observed in parallel with improved mechanical properties, how composition, organization, and mechanical function are related is not known. Moreover, how this relationship compares to that of native fibrocartilage is unclear. Therefore, in the present work, functional fibrocartilage constructs were formed from MSC-seeded nanofibrous scaffolds, and the roles of collagen and glycosaminoglycan (GAG) in compressive and tensile properties were determined. MSCs deposited abundant collagen and GAG over 120 days of culture, and these extracellular molecules were organized in such a way that they performed similar mechanical functions to their native roles: collagen dominated the tensile response while GAG was important for compressive properties. GAG removal resulted in significant stiffening in tension. A similar stiffening response was observed when GAG was removed from native inner annulus fibrosus, suggesting an interaction between collagen fibers and their surrounding extrafibrillar matrix that is shared by both engineered and native fibrocartilages. These findings strongly support the use of electrospun scaffolds and MSCs for fibrocartilage tissue engineering, and provide insight on the structure-function relations of both engineered and native biomaterials.     

Beta type Ti-Mo alloys with changeable Young's modulus for spinal fixation applications

To develop a novel biomedical titanium alloy with a changeable Young's modulus via deformation-induced ω phase transformation for the spinal rods in spinal fixation devices, a series of metastable β type binary Ti-(15-18)Mo alloys were prepared. In this study, the microstructures, Young's moduli and tensile properties of the alloys were systemically examined to investigate the effects of deformation-induced ω phase transformation on their mechanical properties. The springback of the optimal alloy was also examined. Ti-(15-18)Mo alloys subjected to solution treatment comprise a β phase and a small amount of athermal ω phase, and they have low Young's moduli. All the alloys investigated in this study show an increase in the Young's modulus owing to deformation-induced ω phase transformation during cold rolling. The deformation-induced ω phase transformation is accompanied with {332}(β) mechanical twinning. This resulted in the maintenance of acceptable ductility with relatively high strength. Among the examined alloys, the Ti-17Mo alloy shows the lowest Young's modulus and the largest increase in the Young's modulus. This alloy exhibits small springback and could be easily bent to the required shape during operation. Thus, Ti-17Mo alloy is considered to be a potential candidate for the spinal rods in spinal fixation devices.     

Combined use of chondroitinase-ABC,TGF-β1, and collagen crosslinking agent lysyl oxidase to engineer functional neotissues for fibrocartilage repair

Patients suffering from damaged or diseased fibrocartilages currently have no effective long-term treatment options. Despite their potential, engineered tissues suffer from inferior biomechanical integrity and an inability to integrate in vivo. The present study identifies a treatment regimen (including the biophysical agent chondroitinase-ABC, the biochemical agent TGF-β1, and the collagen crosslinking agent lysyl oxidase) to prime highly cellularized, scaffold-free neofibrocartilage implants, effecting continued improvement in vivo. We show these agents drive in vitro neofibrocartilage matrix maturation toward synergistically enhanced Young's modulus and ultimate tensile strength values, which were increased 245% and 186%, respectively, over controls. Furthermore, an in vitro fibrocartilage defect model found this treatment regimen to significantly increase the integration tensile properties between treated neofibrocartilage and native tissue. Through translating this technology to an in vivo fibrocartilage defect model, our results indicate, for the first time, that a pre-treatment can prime neofibrocartilage for significantly enhanced integration potential in vivo, with interfacial tensile stiffness and strength increasing by 730% and 745%, respectively, compared to integration values achieved in vitro. Our results suggest that specifically targeting collagen assembly and organization is a powerful means to augment overall neotissue mechanics and integration potential toward improved clinical feasibility.     

Elastogenic inductability of smooth muscle cells from a rat model of late stage abdominal aortic aneurysms

Although abdominal aortic aneurysms (AAA) can be potentially stabilized by inhibiting inflammatory cell recruitment and their release of proteolytic enzymes, active AAA regression is not possible without regeneration of new elastic matrix structures. Unfortunately, postneonatal vascular smooth muscle cells (SMCs), healthy, and likely more so, diseased cells, poorly synthesize or remodel elastic fibers, impeding any effort directed at regenerative AAA treatment. Previously, we determined the eleastogenic benefits of oligomers (HA-o; 4-6 mers) of the glycosaminoglycan, hyaluronan (HA) and transforming growth factor-β1 (TGF-β1) to healthy SMCs. Since AAAs are often diagnosed only late in development when matrix disruption is severe, we now determine if elastogenic upregulation of SMCs from late-stage AAAs (>100% diameter increase) is possible. AAAs were induced by perfusion of rat infrarenal aortae with porcine pancreatic elastase. Elastic matrix degradation, vessel expansion (～120%), inflammatory cell infiltration, and enhanced activity of matrix-metalloproteases (MMPs) 2 and 9 resulted, paralleling human AAAs. Aneurysmal SMCs (EaRASMCs) maintained a diseased phenotype in 2D cell culture and exhibited patterns of gene expression different from healthy rat aortic SMCs (RASMCs). Relative to passage-matched healthy RASMCs, unstimulated EaRASMCs produced far less tropoelastin and matrix elastin. Exogenous TGF-β and HA-o (termed "factors") significantly decreased EaRASMC proliferation and enhanced tropoelastin synthesis, though only at the highest provided dose combination (20?mg/mL of HA-o, 10?ng/mL of TGF-β); despite such enhancement, tropoelastin amounts were only ～40% of amounts synthesized by healthy RASMC cultures. Differently, elastic matrix synthesis was enhanced beyond amounts synthesized by healthy RASMCs (112%), even at lower doses of factors (2?mg/mL of HA-o and 5?ng/mL of TGF-β). The factors also enhanced elastic fiber deposition over untreated EaRASMC cultures and restored several genes whose expression was altered in EaRASMC cultures back to levels expressed by healthy RASMCs. However, the activity of MMPs 2 and 9 generated by EaRASMC cultures was unaffected by the factors/factor dose. The study confirms that SMCs from advanced AAAs can be elastogenically induced, although much higher doses of elastogenic factors are required for induction relative to healthy SMCs. Also, the factors do not appear to inhibit MMP activity, vital to preserve existing elastic matrix structures that serve as nucleation sites for new elastic fiber deposition. Thus, to enhance net accumulation of newly regenerated elastic matrix, toward possibly regressing AAAs, codelivery of MMP inhibitors may be necessitated.     

Dermis isolated adult stem cells for cartilage tissue engineering

Adult stem cells from the dermal layer of skin are an attractive alternative to primary cells for meniscus engineering, as they may be easily obtained and used autologously. Recently, chondroinducible dermis cells from caprine skin have shown promising characteristics for cartilage tissue engineering. In this study, their multilineage differentiation capacity is determined, and methods of expanding and tissue engineering these cells are investigated. It was found that these cells could differentiate along adipogenic, osteogenic, and chondrogenic lineages, allowing them to be termed dermis isolated adult stem cells (DIAS cells). Focusing on cartilage tissue engineering, it was found that passaging these cells in chondrogenic medium and forming them into self-assembled tissue engineered constructs caused upregulation of collagen type II and COMP gene expression. Further investigation showed that applying transforming growth factor β1 (TGF-β1) or bone morphogenetic protein 2 (BMP-2) to DIAS constructs caused increased sulfated glycosaminoglycan content. Additionally, TGF-β1 treatment caused significant increases in compressive properties and construct contraction. In contrast, BMP-2 treatment resulted in the largest constructs, but did not increase compressive properties. These results show that DIAS cells can be easily manipulated for cartilage tissue engineering strategies, and may also be a useful cell source for other mesenchymal tissues.     

Engineering functional anisotropy in fibrocartilage neotissues

The knee meniscus, intervertebral disc, and temporomandibular joint (TMJ) disc all possess complex geometric shapes and anisotropic matrix organization. While these characteristics are imperative for proper tissue function, they are seldom recapitulated following injury or disease. Thus, this study's objective was to engineer fibrocartilages that capture both gross and molecular structural features of native tissues. Self-assembled TMJ discs were selected as the model system, as the disc exhibits a unique biconcave shape and functional anisotropy. To drive anisotropy, 50:50 co-cultures of meniscus cells and articular chondrocytes were grown in biconcave, TMJ-shaped molds and treated with two exogenous stimuli: biomechanical (BM) stimulation via passive axial compression and bioactive agent (BA) stimulation via chondroitinase-ABC and transforming growth factor-β1. BM + BA synergistically increased Col/WW, Young's modulus, and ultimate tensile strength 5.8-fold, 14.7-fold, and 13.8-fold that of controls, respectively; it also promoted collagen fibril alignment akin to native tissue. Finite element analysis found BM stimulation to create direction-dependent strains within the neotissue, suggesting shape plays an essential role toward driving in vitro anisotropic neotissue development. Methods used in this study offer insight on the ability to achieve physiologic anisotropy in biomaterials through the strategic application of spatial, biomechanical, and biochemical cues.