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Department
Osteointegration of a Novel Silk Fiber-Based ACL Scaffold by Formation of a Ligament-Bone Interface.
(2019)
Effect of fluid dynamics on decellularization efficacy and mechanical properties of blood vessels.
(2019)
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a93591
27-SY-1 Regenerative silk ligament: scale up and regulatory strategy of a textile engineered silk implant for tissue
regeneration of injured human ACL (anterior cruciate ligament)
Bernhard Küenburg,Andreas Teuschl
MorphoMed, Austria
In the USA around 370.000 annual ACL ruptures in the predominately young population (age 18-30) following sport injuries cause a lot of pain
and long term troubles for the affected patients. The well established standard procedure of autologous tissue transplantation stabilizes the
knee and allows sport activities after 9-12 months. However, harvest of autologous tissue such as patellar tendon or semitendinosus (the most
frequently used autograft) weakens other body areas and long term data show an increased rate of arthroses (up to 50% after 10 years)
associated with ACL reconstructions.
Based on numerous preclinical data and data from a 12-month sheep study the scale-up as well as regulatory approval process of a novel
textile engineered silk implant as ACL graft have been initiated. It is planned to enter a clinical trial and strive for regulatory approval as a
medical class 3 implant. The sheep data (1) have revealed an approximately 50% degradation of the purified medical silk, which acts as
scaffold for the regrowth of a new endogenous ligament. The formation of oriented collagenous tissue fibers including vascularization proves a
ligament tissue regeneration for the first time.
Scale-up and implementation of a commercial process require a defined set of specifications of commercial silkworm (Bombyx mori) silk, a
biological raw material sourced from the textile industry as well as adequate analytical methods to characterize the depletion of sericin in the
course of the production process.
Based on first full scale samples, the defined biocompatibility program has to be executed, in the EU following the ISO requirements of the
notified bodies, in the USA determined by the FDA. Upon submission of the full technical documentation as well as the biocompati-bility data,
approval of a clinical trial can be achieved in order to demonstrate the clinical efficacy of the silk based ACL graft compared to the gold
standard method in two different randomized groups. As primary endpoint the knee stability will be tested by an apparatus supported
Lachmann test. In this study, besides the clinical benefit the patient safety is an important goal. The results of this study will be used for
achievement of regulatory approval.
References:
1) Teuschl A, Heimel P, Nürnberger S, van Griensven M, Redl H, Nau T. Am. J. Sports Med. 2016;44:1547–57. 2) Teuschl
AH, van Griensven M, Redl H. Tissue Eng. Part C Methods. 2014;20:431–9.
Inflammation processes are associated with significant decreases in tissue or lysosomal pH from 7.4 to 4, a fact that argues for the application of pH-responsive drug delivery systems. However, for their design and optimization a full understanding of the release mechanism is crucial. In this study we investigated the pH-depending drug release mechanism and the influence of silk fibroin (SF) concentration and SF degradation degree of human serum albumin (HSA)-SF nanocapsules. Sonochemically produced nanocapsules were investigated regarding particle size, colloidal stability, protein encapsulation, thermal stability and drug loading properties. Particles of the monodisperse phase showed average hydrodynamic radii between 438 and 888 nm as measured by DLS and AFM and a zeta potential of -11.12 ± 3.27 mV. Together with DSC results this indicated the successful production of stable nanocapsules. ATR-FTIR analysis demonstrated that SF had a positive effect on particle formation and stability due to induced beta-sheet formation and enhanced crosslinking. The pH-responsive release was found to depend on the SF concentration. In in-vitro release studies, HSA-SF nanocapsules composed of 50% SF showed an increased pH-responsive release for all tested model substances (Rhodamine B, Crystal Violet and Evans Blue) and methotrexate at the lowered pH of 4.5 to pH 5.4, while HSA capsules without SF did not show any pH-responsive drug release. Mechanistic studies using confocal laser scanning microscopy (CLSM) and small angle X-ray scattering (SAXS) analyses showed that increases in particle porosity and decreases in particle densities are directly linked to pH-responsive release properties. Therefore, the pH-responsive release mechanism was identified as diffusion controlled in a novel and unique approach by linking scattering results with in vitro studies. Finally, cytotoxicity studies using the human monocytic THP-1 cell line indicated non-toxic behavior of the drug loaded nanocapsules when applied in a concentration of 62.5 µg mL-1.
There is continual demand for animal models that allow a quantitative assessment of angiogenic properties of biomaterials, therapies, and pharmaceuticals. In its simplest form, this is done by subcutaneous material implantation and subsequent vessel counting which usually omits spatial data. We have refined an implantation model and paired it with a computational analytic routine which outputs not only vessel count but also vessel density, distribution, and vessel penetration depth, that relies on a centric vessel as a reference point. We have successfully validated our model by characterizing the angiogenic potential of a fibrin matrix in conjunction with recombinant human vascular endothelial growth factor (rhVEGF165). The inferior epigastric vascular pedicles of rats were sheathed with silicone tubes, which were subsequently filled with 0.2 ml of fibrin and different doses of rhVEGF165, centrically embedding the vessels. Over 4 weeks, tissue samples were harvested and subsequently immunohistologically stained and computationally analyzed. The model was able to detect variations over the angiogenic potentials of growth factor spiked fibrin matrices. Adding 20 ng of rhVEGF165 resulted in a significant increase in vasculature while 200 ng of rhVEGF165 did not improve vascular growth. Vascularized tissue volume increased during the first week and vascular density increased during the second week. Total vessel count increased significantly and exhibited a peak after 2 weeks which was followed by a resorption of vasculature by week 4. In summary, a simple implantation model to study in vivo vascularization with only a minimal workload attached was enhanced to include morphologic data of the emerging vascular tree.
Treatment of peripheral nerve injuries has evolved over the past several decades to include the use of sophisticated new materials endowed with trophic and topographical cues that are essential for in vivo nerve fibre regeneration. In this research, we explored the use of an advanced design strategy for peripheral nerve repair, using biological and semi-synthetic hydrogels that enable controlled environmental stimuli to regenerate neurons and glial cells in a rat sciatic nerve resection model. The provisional nerve growth conduits were composed of either natural fibrin or adducts of synthetic polyethylene glycol and fibrinogen or gelatin. A photo-patterning technique was further applied to these 3D hydrogel biomaterials, in the form of laser-ablated microchannels, to provide contact guidance for unidirectional growth following sciatic nerve injury. We tested the regeneration capacity of subcritical nerve gap injuries in rats treated with photo-patterned materials and compared these with injuries treated with unpatterned hydrogels, either stiff or compliant. Among the factors tested were shear modulus, biological composition, and micropatterning of the materials. The microchannel guidance patterns, combined with appropriately matched degradation and stiffness properties of the material, proved most essential for the uniform tissue propagation during the nerve regeneration process.
Low revision rate and excellent outcome of primary ACL repair with a minimum follow-up of 5 years.
(2018)
Introduction: Due to limitations of ACL reconstruction, primary ACL repair has recently regained research interest. Although abandoned in the past, primary repair with conservation of the original ligament demonstrates considerable advantages compared to reconstruction.
We hypothesized that early repair, strictly limited to patients with a proximal ACL rupture and excellent tissue quality of the remaining ACL stump, would lead to equal revision rates and subjective outcomes as reported for ACL reconstruction after a minimum of 5 years.
Methods: In this questionnaire study, patients who had a primary ACL repair between 2002 and 2009 were invited to participate. Besides any potential revision surgery, the Tegner activity scale and the Knee Injury and Osteoarthritis Outcome Score were included in the evaluation.
Results: Out of 1912 patients who had ACL related surgery during the observation period, 221 (11.4%) had a primary ACL repair. 60 patients (61 knees) were available for follow-up. In 2/61 (3.3%) cases ACL revision surgery was performed and one patient had meniscus surgery of the affected side. The median Tegner activity scale was 6 (range, 3 to 10). The mean KOOS subscores were 88.8% (Function/Sports), 86.6% (Quality of life), 94.6 (Symptoms), 94.0 (Pain) and 97.0 (Activities of Daily Living).
Conclusion: Primary ACL repair, strictly limited to proximal ruptures with good tissue quality leads to revision rates and subjective outcome comparable to ACL reconstruction. Level of evidence: IV.
Extracorporeal shockwave treatment: A novel tool to improve Schwann cell isolation and culture
(2015)
Bioreactor and scaffold design for the mechanical stimulation of anterior cruciate ligament grafts
(2013)
Articular cartilage tissue regeneration: current research strategies and outlook for the future
(2013)
Silk fibroin based carrier system for delivery of fibrinogen and thrombin as coagulant supplements
(2016)
Small diameter vascular grafts from human placenta, decellularized with either Triton X-100 (Triton) or SDS and crosslinked with heparin were constructed and characterized. Graft biochemical properties, residual DNA, and protein composition were evaluated to compare the effect of the two detergents on graft matrix composition and structural alterations. Biocompatibility was tested in vitro by culturing the grafts with primary human macrophages and in vivo by subcutaneous implantation of graft conduits (n = 7 per group) into the flanks of nude rats. Subsequently, graft performance was evaluated using an aortic implantation model in Sprague Dawley rats (one month, n = 14). In situ graft imaging was performed using MRI angiography. Retrieved specimens were analyzed by electromyography, scanning electron microscopy, histology and immunohistochemistry to evaluate cell migration and the degree of functional tissue remodeling. Both decellularization methods resulted in grafts of excellent biocompatibility in vitro and in vivo, with low immunogenic potential. Proteomic data revealed removal of cytoplasmic proteins with relative enrichment of ECM proteins in decelluarized specimens of both groups. Noteworthy, LC-Mass Spectrometry analysis revealed that 16 proteins were exclusively preserved in Triton decellularized specimens in comparison to SDS-treated specimens. Aortic grafts showed high patency rates, no signs of thrombus formation, aneurysms or rupture. Conduits of both groups revealed tissue-specific cell migration indicative of functional remodeling. This study strongly suggests that decellularized allogenic grafts from the human placenta have the potential to be used as vascular replacement materials. Both detergents produced grafts with low residual immunogenicity and appropriate mechanical properties. Observed differences in graft characteristics due to preservation method had no impact on successful in vivo performance in the rodent model.
Repopulation of an auricular cartilage scaffold, AuriScaff, perforated with an enzyme combination
(2019)
Biomaterials currently in use for articular cartilage regeneration do not mimic the composition or architecture of hyaline cartilage, leading to the formation of repair tissue with inferior characteristics. In this study we demonstrate the use of "AuriScaff", an enzymatically perforated bovine auricular cartilage scaffold, as a novel biomaterial for repopulation with regenerative cells and for the formation of high-quality hyaline cartilage. AuriScaff features a traversing channel network, generated by selective depletion of elastic fibers, enabling uniform repopulation with therapeutic cells. The complex collagen type II matrix is left intact, as observed by immunohistochemistry, SEM and TEM. The compressive modulus is diminished, but three times higher than in the clinically used collagen type I/III scaffold that served as control. Seeding tests with human articular chondrocytes (hAC) alone and in co-culture with human adipose-derived stromal/stem cells (ASC) confirmed that the network enabled cell migration throughout the scaffold. It also guides collagen alignment along the channels and, due to the generally traverse channel alignment, newly deposited cartilage matrix corresponds with the orientation of collagen within articular cartilage. In an osteochondral plug model, AuriScaff filled the complete defect with compact collagen type II matrix and enabled chondrogenic differentiation inside the channels. Using adult articular chondrocytes from bovine origin (bAC), filling of even deep defects with high-quality hyaline-like cartilage was achieved after 6 weeks in vivo. With its composition and spatial organization, AuriScaff provides an optimal chondrogenic environment for therapeutic cells to treat cartilage defects and is expected to improve long-term outcome by channel-guided repopulation followed by matrix deposition and alignment. STATEMENT OF SIGNIFICANCE: After two decades of tissue engineering for cartilage regeneration, there is still no optimal strategy available to overcome problems such as inconsistent clinical outcome, early and late graft failures. Especially large defects are dependent on biomaterials and their scaffolding, guiding and protective function. Considering the currently used biomaterials, structure and mechanical properties appear to be insufficient to fulfill this task. The novel scaffold developed within this study is the first approach enabling the use of dense cartilage matrix, repopulate it via channels and provide the cells with a compact collagen type II environment. Due to its density, it also provides better mechanical properties than materials currently used in clinics. We therefore think, that the auricular cartilage scaffold (AuriScaff) has a high potential to improve future cartilage regeneration approaches.
Vascular grafts with a diameter of less than 6 mm are made from a variety of materials and techniques to provide alternatives to autologous vascular grafts. Decellularized materials have been proposed as a possible approach to create extracellular matrix (ECM) vascular prostheses as they are naturally derived and inherently support various cell functions. However, these desirable graft characteristics may be limited by alterations of the ECM during the decellularization process leading to decreased biomechanical properties and hemocompatibility. In this study, arteries from the human placenta chorion were decellularized using two distinct detergents (Triton X-100 or SDS), which differently affect ECM ultrastructure. To overcome biomechanical strength loss and collagen fiber exposure after decellularization, riboflavin-mediated UV (RUV) crosslinking was used to uniformly crosslink the collagenous ECM of the grafts. Graft characteristics and biocompatibility with and without RUV crosslinking were studied in vitro and in vivo. RUV-crosslinked ECM grafts showed significantly improved mechanical strength and smoothening of the luminal graft surfaces. Cell seeding using human endothelial cells revealed no cytotoxic effects of the RUV treatment. Short-term aortic implants in rats showed cell migration and differentiation of host cells. Functional graft remodeling was evident in all grafts. Thus, RUV crosslinking is a preferable tool to improve graft characteristics of decellularized matrix conduits.
Repopulation of an auricular cartilage scaffold, AuriScaff, perforated with an enzyme combination.
(2019)
Vascular grafts with a diameter of less than 6 mm are made from a variety of materials and techniques to provide alternatives to autologous vascular grafts. Decellularized materials have been proposed as a possible approach to create extracellular matrix (ECM) vascular prostheses as they are naturally derived and inherently support various cell functions. However, these desirable graft characteristics may be limited by alterations of the ECM during the decellularization process leading to decreased biomechanical properties and hemocompatibility. In this study, arteries from the human placenta chorion were decellularized using two distinct detergents (Triton X-100 or SDS), which differently affect ECM ultrastructure. To overcome biomechanical strength loss and collagen fiber exposure after decellularization, riboflavin-mediated UV (RUV) crosslinking was used to uniformly crosslink the collagenous ECM of the grafts. Graft characteristics and biocompatibility with and without RUV crosslinking were studied in vitro and in vivo. RUV-crosslinked ECM grafts showed significantly improved mechanical strength and smoothening of the luminal graft surfaces. Cell seeding using human endothelial cells revealed no cytotoxic effects of the RUV treatment. Short-term aortic implants in rats showed cell migration and differentiation of host cells. Functional graft remodeling was evident in all grafts. Thus, RUV crosslinking is a preferable tool to improve graft characteristics of decellularized matrix conduits.