A New Bioactive Fibrin Formulation Provided Superior Cartilage Regeneration in a Caprine Model.

The effective and long-term treatment of cartilage defects is an unmet need among patients worldwide. In the past, several synthetic and natural biomaterials have been designed to support functional articular cartilage formation. However, they have mostly failed to enhance the terminal stage of chondrogenic differentiation, leading to scar tissue formation after the operation. Growth factors substantially regulate cartilage regeneration by acting on receptors to trigger intracellular signaling and cell recruitment for tissue regeneration. In this study, we investigated the effect of recombinant insulin-like growth factor 1 (rIGF-1), loaded in fibrin microbeads (FibIGF1), on cartilage regeneration. rIGF-1-loaded fibrin microbeads were injected into full-thickness cartilage defects in the knees of goats. The stability, integration, and quality of tissue repair were evaluated at 1 and 6 months by gross morphology, histology, and collagen type II staining. The in vivo results showed that compared to plain fibrin samples, particularly at 6 months, FibIGF1 improved the functional cartilage formation, confirmed through gross morphology, histology, and collagen type II immunostaining. FibIGF1 could be a promising candidate for cartilage repair in the clinic.

Quantitative intrinsic auto-cathodoluminescence can resolve spectral signatures of tissue-isolated collagen extracellular matrix.

By analyzing isolated collagen gel samples, we demonstrated in situ detection of spectrally deconvoluted auto-cathodoluminescence signatures of specific molecular content with precise spatial localization over a maximum field of view of 300 µm. Correlation of the secondary electron and the hyperspectral images proved ~40 nm resolution in the optical channel, obtained due to a short carrier diffusion length, suppressed by fibril dimensions and poor electrical conductivity specific to their organic composition. By correlating spectrally analyzed auto-cathodoluminescence with mass spectroscopy data, we differentiated spectral signatures of two extracellular matrices, namely human fibrin complex and rat tail collagen isolate, and uncovered differences in protein distributions of isolated extracellular matrix networks of heterogeneous populations. Furthermore, we demonstrated that cathodoluminescence can monitor the progress of a human cell-mediated remodeling process, where human collagenous matrix was deposited within a rat collagenous matrix. The revealed change of the heterogeneous biological composition was confirmed by mass spectroscopy.

Engineered acellular collagen scaffold for endogenous cell guidance, a novel approach in urethral regeneration.

UNLABELLED: The treatment of congenital malformations or injuries of the urethra using existing autologous tissues can be associated with post-operative complications. Using rat-tail collagen, we have engineered an acellular high-density collagen tube. These tubes were made of 2 layers and they could sustain greater burst pressures than the monolayered tubes. Although it remains a weak material this 2 layered tube could be sutured to the native urethra. In 20 male New Zealand white rabbits, 2cm long grafts were sutured in place after subtotal excision of the urethra. This long-term study was performed in Lausanne (Switzerland) and in Kuala Lumpur (Malaysia). No catheter was placed post-operatively. All rabbits survived the surgical implantation. The animals were evaluated at 1, 3, 6, and 9months by contrast voiding cysto-urethrography, histological examination and immunohistochemistry. Spontaneous re-population of urothelial and smooth muscle cells on all grafts was demonstrated. Cellular organization increased with time, however, 20% of both fistula and stenosis could be observed post-operatively. This off-the shelf scaffold with a promising urethral regeneration has a potential for clinical application. STATEMENT OF SIGNIFICANCE: In this study we have tissue engineered a novel cell free tubular collagen based scaffold and used it as a urethral graft in a rabbit model. The novelty of our technique is that the tube can be sutured. Testing showed better burst pressures and the grafts could then be successfully implanted after a urethral excision. This long term study demonstrated excellent biocompatibility of the 2cm graft and gradual regeneration with time, challenging the current literature. Finally, the main impact is that we describe an off-the-shelf and cost-effective product with comparable surgical outcome to the cellular grafts.

Tubular Compressed Collagen Scaffolds for Ureteral Tissue Engineering in a Flow Bioreactor System.

Ureteral replacement by tissue engineering might become necessary following tissue loss after excessive ureteral trauma, after retroperitoneal cancer, or even after failed reconstructive surgery. This need has driven innovation in the design of novel scaffolds and specific cell culture techniques for urinary tract reconstruction. In this study, compressed tubular collagen scaffolds were evaluated, addressing the physical and biological characterization of acellular and cellular collagen tubes in a new flow bioreactor system, imitating the physiological pressure, peristalsis, and flow conditions of the human ureter. Collagen tubes, containing primary human smooth muscle and urothelial cells, were evaluated regarding their change in gene and protein expression under dynamic culture conditions. A maximum intraluminal pressure of 22.43 ± 0.2 cm H2O was observed in acellular tubes, resulting in a mean wall shear stress of 4 dynes/cm(2) in the tubular constructs. Dynamic conditions directed the differentiation of both cell types into their mature forms. This was confirmed by their gene expression of smooth muscle alpha-actin, smoothelin, collagen type I and III, elastin, laminin type 1 and 5, cytokeratin 8, and uroplakin 2. In addition, smooth muscle cell alignment predominantly perpendicular to the flow direction was observed, comparable to the cell orientation in native ureteral tissue. These results revealed that coculturing human smooth muscle and urothelial cells in compressed collagen tubes under human ureteral flow-mimicking conditions could lead to cell-engineered biomaterials that might ultimately be translated into clinical applications.

A collagen-poly(lactic acid-co-ɛ-caprolactone) hybrid scaffold for bladder tissue regeneration.

Scaffold materials should favor cell attachment and proliferation, and provide designable 3D structures with appropriate mechanical strength. Collagen matrices have proven to be beneficial scaffolds for tissue regeneration. However, apart from small intestinal submucosa, they offer a limited mechanical strength even if crosslinking can enhance their mechanical properties. A more cell-friendly way to increase material strength is to combine synthetic polymer meshes with plastic compressed collagen gels. This work describes the potential of plastic compressed collagen-poly(lactic acid-co-ɛ-caprolactone) (PLAC) hybrids as scaffolds for bladder tissue regeneration. Human bladder smooth muscle and urothelial cells were cultured on and inside collagen-PLAC hybrids in vitro. Scaffolds were analyzed by electron microscopy, histology, immunohistochemistry, and AlamarBlue assay. Both cell types proliferated in and on the hybrid, forming dense cell layers on top after two weeks. Furthermore, hybrids were implanted subcutaneously in the backs of nude mice. Host cell infiltration, scaffold degradation, and the presence of the seeded bladder cells were analyzed. Hybrids showed a lower inflammatory reaction in vivo than PLAC meshes alone, and first signs of polymer degradation were visible at six months. Collagen-PLAC hybrids have potential for bladder tissue regeneration, as they show efficient cell seeding, proliferation, and good mechanical properties.

High-density collagen gel tubes as a matrix for primary human bladder smooth muscle cells.

Tissue-engineered grafts for the urinary tract are being investigated for the potential treatment of several urologic diseases. These grafts, predominantly tubular-shaped, usually require in vitro culture prior to implantation to allow cell engraftment on initially cell-free scaffolds. We have developed a method to produce tubular-shaped collagen scaffolds based on plastic compression. Our approach produces a ready cell-seeded graft that does not need further in vitro culture prior to implantation. The tubular collagen scaffolds were in particular investigated for their structural, mechanical and biological properties. The resulting construct showed an especially high collagen density, and was characterized by favorable mechanical properties assessed by axial extension and radial dilation. Young modulus in particular was greater than non-compressed collagen tubes. Seeding densities affected proliferation rate of primary human bladder smooth muscle cells. An optimal seeding density of 10(6) cells per construct resulted in a 25-fold increase in Alamar blue-based fluorescence after 2 wk in culture. These high-density collagen gel tubes, ready seeded with smooth muscle cells could be further seeded with urothelial cells, drastically shortening the production time of graft for urinary tract regeneration.

In vivo study of an injectable poly(acrylonitrile)-based hydrogel paste as a bulking agent for the treatment of urinary incontinence.

Urinary incontinence can be treated by endoscopic injection of bulking agents, however, no optimal therapeutic effect has been achieved upon this treatment yet. In the present study, the development of a injectable poly(acrylonitrile) hydrogel paste is described, and its efficacy and histological behavior, once injected into the submucosal space of the minipig bladder, are evaluated. A device was developed to mix poly(acrylonitrile) hydrogel powder with glycerin, used as carrier, prior to injection into the submucosal space of the bladder. Several paste deposits, depending on the size of the bladder, were injected per animal. The implants were harvested at days 7, 14, 21, 28, 84 and 168 and analyzed morphologically and by histology. The persistence of the implants was demonstrated. However, at later time points the implants were split up and surrounded by granulomatous tissue, which was gradually replaced by histiocytes and adipocytes. Transitory focal urothelial metaplasia was observed only at day 7 and moderate foreign body reaction was detected predominantly between the second and fifth week. This study demonstrated the feasibility to develop an injectable paste of poly(acrylonitrile) hydrogel thought to provide the expected bulking effect, necessary for the treatment of urinary incontinence.