Frequently Used Strategies to Isolate Extracellular Matrix Proteins from Human Placenta and Adipose Tissue.

The natural extracellular matrix (ECM) provides the optimal environment for cells. Many enzymatic or non-enzymatic based strategies to extract ECM proteins from tissues were published over the past years. However, every single isolation strategy reported so far is associated with specific bottlenecks. In this study, frequently used strategies to isolate ECM from human placenta or adipose tissue using Tris-, serum-, or pepsin-based buffers were compared. The resulting ECM proteins were biochemically characterized by analysis of cellular remnants using Hoechst DNA staining, glycosaminoglycan (GAG) content by dimethylmethylene blue, visualization of protein bands using sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis combined with amino acid quantification, and assessment of the proangiogenic profile using an angiogenesis array. Tris-NaCl-extracted ECM proteins showed a high heterogenic degree of extracted proteins, bioactive growth factors, and GAGs, but no collagen-I. Active serum-extracted ECM showed significant lower DNA remnants when compared with the Tris-NaCl isolation strategy. Pepsin-extracted ECM was rich in collagen-I and low amounts of remaining bioactive growth factors. This strategy was most effective to reduce DNA amounts when compared with the other isolation strategies. Pepsin-extracted ECM from both tissues easily gelled at 37°C, whereas the other extracted ECM strategies did not gel at 37°C (Tris-NaCl: liquid; serum: sponge). All relevant characteristics (DNA residues, ECM diversity and bioactivity, shape) of the extracted ECM proteins highly depend on its isolation strategy and could still be optimized. Impact statement The natural human extracellular matrix (ECM) is the ideal cell niche. Various strategies were reported to isolate human ECM components from various sources. In this article, we compared frequently used methods and compared their characteristics (DNA remnants, glycosaminoglycan content, sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis, amino acid quantification, angiogenesis array, and gel formation). We conclude that more research is still necessary to optimize current isolation approaches for in vitro or in vivo applications of human ECM.

Crosslinking strategies for silk fibroin hydrogels: promising biomedical materials.

Due to their strong biomimetic potential, silk fibroin (SF) hydrogels are impressive candidates for tissue engineering, due to their tunable mechanical properties, biocompatibility, low immunotoxicity, controllable biodegradability, and a remarkable capacity for biomaterial modification and the realization of a specific molecular structure. The fundamental chemical and physical structure of SF allows its structure to be altered using various crosslinking strategies. The established crosslinking methods enable the formation of three-dimensional (3D) networks under physiological conditions. There are different chemical and physical crosslinking mechanisms available for the generation of SF hydrogels (SFHs). These methods, either chemical or physical, change the structure of SF and improve its mechanical stability, although each method has its advantages and disadvantages. While chemical crosslinking agents guarantee the mechanical strength of SFH through the generation of covalent bonds, they could cause some toxicity, and their usage is not compatible with a cell-friendly technology. On the other hand, physical crosslinking approaches have been implemented in the absence of chemical solvents by the induction of ß-sheet conformation in the SF structure. Unfortunately, it is not easy to control the shape and properties of SFHs when using this method. The current review discusses the different crosslinking mechanisms of SFH in detail, in order to support the development of engineered SFHs for biomedical applications.

Iodine-Enhanced Micro-CT Imaging of Soft Tissue on the Example of Peripheral Nerve Regeneration.

Microcomputed tomography (µCT) is widely used for the study of mineralized tissues, but a similar use for soft tissues is hindered by their low X-ray attenuation. This limitation can be overcome by the recent development of different staining techniques. Staining with Lugol's solution, a mixture of one part iodine and two parts potassium iodide in water, stands out among these techniques for its low complexity and cost. Currently, Lugol staining is mostly used for anatomical examination of tissues. In the present study, we seek to optimize the quality and reproducibility of the staining for ex vivo visualization of soft tissues in the context of a peripheral nerve regeneration model in the rat. We show that the staining result not only depends on the concentration of the staining solution but also on the amount of stain in relation to the tissue volume and composition, necessitating careful adaptation of the staining protocol to the respective specimen tissue. This optimization can be simplified by a stepwise staining which we show to yield a similar result compared to staining in a single step. Lugol staining solution results in concentration-dependent tissue shrinkage which can be minimized but not eliminated. We compared the shrinkage of tendon, nerve, skeletal muscle, heart, brain, and kidney with six iterations of Lugol staining. 60 ml of 0.3% Lugol's solution per cm3 of tissue for 24 h yielded good results on the example of a peripheral nerve regeneration model, and we were able to show that the regenerating nerve inside a silk fibroin tube can be visualized in 3D using this staining technique. This information helps in deciding the region of interest for histological imaging and provides a 3D context to histological findings. Correlating both imaging modalities has the potential to improve the understanding of the regenerative process.

Osteointegration of a Novel Silk Fiber-Based ACL Scaffold by Formation of a Ligament-Bone Interface.

BACKGROUND: Given the unsatisfactory results and reported drawbacks of anterior cruciate ligament (ACL) reconstruction, such as donor site morbidity and the limited choice of grafts in revision surgery, new regenerative approaches based on tissue-engineering strategies are currently under investigation. PURPOSES: To determine (1) if a novel silk fiber-based ACL scaffold is able to initiate osteointegration in the femoral and tibial bone tunnels under in vivo conditions and (2) if the osteointegration process will be improved by intraoperatively seeding the scaffolds with the autologous stromal vascular fraction, an adipose-derived, stem cell-rich isolate from knee fat pads. STUDY DESIGN: Controlled laboratory study. METHODS: A total of 33 sheep underwent ACL resection and were then randomly assigned to 2 experimental groups: ACL reconstruction with a scaffold alone and ACL reconstruction with a cell-seeded scaffold. Half of the sheep in each group were randomly chosen and euthanized 6 months after surgery and the other half at 12 months. To analyze the integration of the silk-based scaffold in the femoral and tibial bone tunnels, hard tissue histology and micro-computed tomography measurements were performed. RESULTS: Hard tissue histological workup showed that in all treatment groups, with or without the application of the autologous stromal vascular fraction, an interzone of collagen fibers had formed between bone and silk-based graft. This collagen-fiber continuity partly consisted of Sharpey fibers, comparable with tendon-bone healing known for autografts and allografts. Insertion sites were more broad based at 6 months and more concentrated on the slightly protruding, bony knoblike structures at 12 months. Histologically, no differences between the treatment groups were detectable. Analysis of micro-computed tomography measurements revealed a significantly higher tissue density for the cell-seeded scaffold group as compared with the scaffold-alone group in the tibial but not femoral bone tunnel after 12 months of implantation. CONCLUSION: The novel silk fiber-based scaffold for ACL regeneration demonstrated integration into the bone tunnels via the formation of a fibrous interzone similar to allografts and autografts. Histologically, additional cell seeding did not enhance osteointegration. No significant differences between 6 and 12 months could be detected. After 12 months, there was still a considerable amount of silk present, and a longer observation period is necessary to see if a true ligament-bone enthesis will be formed. CLINICAL RELEVANCE: ACL regeneration with a silk fiber-based scaffold with and without additional cell seeding may provide an alternative treatment option to current techniques of surgical reconstruction.

A Low Cost Implantation Model in the Rat That Allows a Spatial Assessment of Angiogenesis.

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.

Decellularized human placenta chorion matrix as a favorable source of small-diameter vascular grafts.

Biomaterials based on decellularized tissues are increasingly attracting attention as functional alternatives to other natural or synthetic materials. However, a source of non-cadaver human allograft material would be favorable. Here we establish a decellularization method of vascular tissue from cryopreserved human placenta chorionic plate starting with an initial freeze-thaw step followed by a series of chemical treatments applied with a custom-made perfusion system. This novel pulsatile perfusion set-up enabled us to successfully decellularize the vascular tissue with lower concentrations of chemicals and shorter exposure times compared to a non-perfusion process. The decellularization procedure described here lead to the preservation of the native extracellular matrix architecture and the removal of cells. Quantitative analysis revealed no significant changes in collagen content and a retained glycosaminoglycan content of approximately 29%. In strain-to-failure tests, the decellularized grafts showed similar mechanical behavior compared to native controls. In addition, the mechanical values for ultimate tensile strength and stiffness were in an acceptable range for in vivo applications. Furthermore, biocompatibility of the decellularized tissue and its recellularizationability to serve as an adequate substratum for upcoming recellularization strategies using primary human umbilical vein endothelial cells (HUVECs) was demonstrated. HUVECs cultured on the decellularized placenta vessel matrix performed endothelialization and maintained phenotypical characteristics and cell specific expression patterns. Overall, the decellularized human placenta vessels can be a versatile tool for experimental studies on vascularization and as potent graft material for future in vivo applications. STATEMENT OF SIGNIFICANCE: In the US alone more than 1million vascular grafts are needed in clinical practice every year. Despite severe disadvantages, such as donor site morbidity, autologous grafting from the patient's own arteries or veins is regarded as the gold standard for vascular tissue repair. Besides, strategies based on synthetic or natural materials have shown limited success. Tissue engineering approaches based on decellularized tissues are regarded as a promising alternative to clinically used treatments to overcome the observed limitations. However, a source for supply of non-cadaver human allograft material would be favorable. Here, we established a decellularization method of vascular tissue from the human placenta chorionic plate, a suitable human tissue source of consistent quality. The decellularized human placenta vessels can be a potent graft material for future in vivo applications and furthermore might be a versatile tool for experimental studies on vascularization.

A novel bioreactor for the generation of highly aligned 3D skeletal muscle-like constructs through orientation of fibrin via application of static strain.

The generation of functional biomimetic skeletal muscle constructs is still one of the fundamental challenges in skeletal muscle tissue engineering. With the notion that structure strongly dictates functional capabilities, a myriad of cell types, scaffold materials and stimulation strategies have been combined. To further optimize muscle engineered constructs, we have developed a novel bioreactor system (MagneTissue) for rapid engineering of skeletal muscle-like constructs with the aim to resemble native muscle in terms of structure, gene expression profile and maturity. Myoblasts embedded in fibrin, a natural hydrogel that serves as extracellular matrix, are subjected to mechanical stimulation via magnetic force transmission. We identify static mechanical strain as a trigger for cellular alignment concomitant with the orientation of the scaffold into highly organized fibrin fibrils. This ultimately yields myotubes with a more mature phenotype in terms of sarcomeric patterning, diameter and length. On the molecular level, a faster progression of the myogenic gene expression program is evident as myogenic determination markers MyoD and Myogenin as well as the Ca(2+) dependent contractile structural marker TnnT1 are significantly upregulated when strain is applied. The major advantage of the MagneTissue bioreactor system is that the generated tension is not exclusively relying on the strain generated by the cells themselves in response to scaffold anchoring but its ability to subject the constructs to individually adjustable strain protocols. In future work, this will allow applying mechanical stimulation with different strain regimes in the maturation process of tissue engineered constructs and elucidating the role of mechanotransduction in myogenesis. STATEMENT OF SIGNIFICANCE: Mechanical stimulation of tissue engineered skeletal muscle constructs is a promising approach to increase tissue functionality. We have developed a novel bioreactor-based 3D culture system, giving the user the possibility to apply different strain regimes like static, cyclic or ramp strain to myogenic precursor cells embedded in a fibrin scaffold. Application of static mechanical strain leads to alignment of fibrin fibrils along the axis of strain and concomitantly to highly aligned myotube formation. Additionally, the pattern of myogenic gene expression follows the temporal progression observed in vivo with a more thorough induction of the myogenic program when static strain is applied. Ultimately, the strain protocol used in this study results in a higher degree of muscle maturity demonstrated by enhanced sarcomeric patterning and increased myotube diameter and length. The introduced bioreactor system enables new possibilities in muscle tissue engineering as longer cultivation periods and different strain applications will yield tissue engineered muscle-like constructs with improved characteristics in regard to functionality and biomimicry.

Emerging Trends in Abdominal Wall Reinforcement: Bringing Bio-Functionality to Meshes.

Abdominal wall hernia is a recurrent issue world-wide and requires the implantation of over 1 million meshes per year. Because permanent meshes such as polypropylene and polyester are not free of complications after implantation, many mesh modifications and new functionalities have been investigated over the last decade. Indeed, mesh optimization is the focus of intense development and the biomaterials utilized are now envisioned as being bioactive substrates that trigger various physiological processes in order to prevent complications and to promote tissue integration. In this context, it is of paramount interest to review the most relevant bio-functionalities being brought to new meshes and to open new avenues for the innovative development of the next generation of meshes with enhanced properties for functional abdominal wall hernia repair.

A New Preparation Method for Anisotropic Silk Fibroin Nerve Guidance Conduits and Its Evaluation In Vitro and in a Rat Sciatic Nerve Defect Model.

Over the past decade, silk fibroin (SF) has been emergently used in peripheral nerve tissue engineering. Current approaches aiming at producing SF-based nerve guidance conduits (SF-NGCs) used dissolved silk based on either aqueous solutions or organic solvents. In this study, we describe a novel procedure to produce SF-NGCs: A braided tubular structure of raw Bombyx mori silk is subsequently processed with the ternary solvent CaCl2/H2O/ethanol, formic acid, and methanol to improve its mechanical and topographical characteristics. Topographically, the combination of the treatments results in a fusion of the outer single silk fibers to a closed layer with a thickness ranging from about 40 to 75 µm. In contrast to the outer wall, the inner lumen (not treated with processing solvents) still represents the braided structure of single fibers. Mechanical stability, elasticity, and kink characteristics were evaluated with a custom-made test system. The modification procedure described here drastically improved the elastic properties of our tubular raw scaffold, favoring its use as a NGC. A cell migration assay with NIH/3T3-fibroblasts revealed the impermeability of the SF-NGC wall for possible invading and scar-forming cells. Moreover, the potential of the SF-NGC to serve as a substratum for Schwann cells has been demonstrated by cytotoxicity tests and live-dead stainings of Schwann cells grown on the inner surface of the SF-NGC. In vivo, the SF-NGC was tested in a rat sciatic nerve injury model. In short-term in vivo studies, it was proved that SF-NGCs are not triggering host inflammatory reactions. After 12 weeks, we could demonstrate morphological and functional reinnervation of the distal targets. Filled with collagen, a higher number of axons could be found in the distal to the graft (1678±303), compared with the empty SF-NGC (1274±146). The novel SF-NGC presented here shows promising results for the treatment of peripheral nerve injuries. The modification of braided structures to adapt their mechanical and topographical characteristics may support the translation of SF-based scaffolds into the clinical setting. However, further improvements and the use of extracellular matrix molecules and Schwann cells are suggested to enable silk tube based conduits to bridge long-distance nerve gaps.

In vitro extracorporeal shock wave treatment enhances stemness and preserves multipotency of rat and human adipose-derived stem cells.

BACKGROUND AIMS: Adipose-derived progenitor/stem cells (ASCs) are discussed as a promising candidate for various tissue engineering approaches. However, its applicability for the clinic is still difficult due to intra- and inter-donor heterogeneity and limited life span in vitro, influencing differentiation capacity as a consequence to decreased multipotency. METHODS: Extracorporeal shock wave treatment has been proven to be a suitable clinical tool to improve regeneration of a variety of tissues for several decades, whereas the mechanisms underlying these beneficial effects remain widely unknown. RESULTS: In this study we show that human and rat adipose derived stem cells respond strongly to repetitive shock wave treatment in vitro, resulting not only in maintenance and significant elevation of mesenchymal markers (CD73, CD90, CD105), but also in significantly increased differentiation capacity towards the osteogenic and adipogenic lineage as well as toward Schwann-cell like cells even after extended time in vitro, preserving multipotency of ASCs. CONCLUSIONS: ESWT might be a promising tool to improve ASC quality for cell therapy in various tissue engineering and regenerative medicine applications.

Sericin removal from raw Bombyx mori silk scaffolds of high hierarchical order.

Silk fibroin has previously been described as a promising candidate for ligament tissue engineering (TE) approaches. For biocompatibility reasons, silkworm silk requires removal of sericin, which can elicit adverse immune responses in the human body. One disadvantage of the required degumming process is the alteration of the silk fiber structural properties, which can hinder textile engineering of high order hierarchical structures. Therefore, the aim of this study was to find a way to remove sericin from a compact and highly ordered raw silk fiber matrix. The wire rope design of the test model scaffold comprises several levels of geometric hierarchy. Commonly used degumming solutions fail in removing sericin in this wire rope design. Weight loss measurements, picric acid and carmine staining as well as scanning electron microscopy demonstrated that the removal of sericin from the model scaffold of a wire rope design can be achieved through a borate buffer-based system. Furthermore, the borate buffer degummed silks were shown to be nontoxic and did not alter cell proliferation behavior. The possibility to remove sericin after the textile engineering process has taken place eases the production of highly ordered scaffold structures and may expand the use of silk as scaffold material in further TE and regenerative medicine applications.