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Hydrogels are common biomaterials that find their application in tissue engineering, and can be used for tissue regeneration. The introduction of a biomaterial into the tissue causes specific cellular outcomes as the cells can not only sense the presence of bioactive ligands but also the stiffness and mechanical properties of the material. The mechanotransduction-based pathways influence the adhesion, migration and differentiation of cells Therefore, one of the benefits of using hydrogel-based scaffolds is that their biomechanical and biochemical properties are highly tunable and can be optimized to mimic a tissue. This means that those hydrogel-based scaffolds have to mimic the extracellular environment, or extracellular matrix (ECM) of the tissues that the cells are normally embedded in. In today’s blog post I will try to present an interesting polymer composite made out of Poly(ethylene glycol) (PEG), and self-assembled peptide amphiphile (PA).

In this design, PEG provides for the mechanical base of the scaffold. The benefits of using this hydrogel are that it is water-soluble, highly biocompatible, and nonimmunogenic. However, its downside is that PEG as such does not allow for cell adhesion and therefore must be functionalized with ECM-derived bioactive proteins. The approach allows for including peptides without affecting the crosslinking of PEG through photoactivation.

PA on the other hand can self-assemble into cylindrical, fibrous networks under physiological pH an temperature. Upon incorporation of bioactive proteins, PA hydrogels become a highly biocompatible, fibrous materials which allow for cell adhesion.

The hydrogel composite presented in the paper I reviewed, consisted of PA with incorporated bioactive peptides, namely RGD, DGEA (from collagen I), and PEG-dimethacrylate (PEGDMA) in different volumetric ratios. That is, the final concentrations of PEGDMA were in the range 4-15% (w/v) and 0.1% (w/v) of the photoinitiator, which allowed for obtaining gels of different Young’s modulus (0.1-8 kPa). The self-assembling PA proteins included a hydrophobic fatty acid and hydrophilic peptide parts which allowed for the self-assembly of PA within the composite, and were further conjugated to the bioactive peptides. The final concentration of PA in the composite was 1.5%, and was enough to allow for cell adhesion, spreading and proliferation. Overall, the design is such that PA-peptide and PEG are mixed under physiological conditions to allow for formation of nanofibers within the solution first, and only then it is subjected to photoactivation and the methacrylate groups of PEGDMA form a polymeric network. As a result, the composite is lamellar, mesoporous (pores in the nanometer range) and resembles the ECM structure. The gels also show isotropic behavior due to the equal dispersion of PA in PEGDMA. The bioactive peptides on PA are available for cells and do not alter the PEG crosslinking. Additionally, this means that the same structure should be obtained regardless of the peptide incorporated into PA, and the structural properties should not be altered.

Why am I trying to draw your attention to ECM-mimetic composites? Throughout this semester I have been trying to find biomaterial-based approaches for the treatment of fibrosis in different tissues and organs, however I have found that the field still offers many opportunities for new advancements. This ECM biomimetic allows for both altering the biochemical composition as well as biomechanical properties of the gels. Both 3D and 2D gels can be constructed with use of this design. This and similar designs could be used in research, which would allow for better understanding of fibrotic diseases and ultimately designing new, hopefully successful treatments.

 

References:

Goktas, M., Cinar, G., Orujalipoor, I., Ide, S., Tekinay, A. B., & Guler, M. O. (2015). Self-assembled peptide amphiphile nanofibers and PEG composite hydrogels as tunable ECM mimetic microenvironment. Biomacromolecules, 16(4), 1247-1258.

Chronic pancreatitis is a result of unresolved and prolonged inflammation of the pancreas and eventually leads to pancreatic fibrosis and obstruction of the pancreatic and biliary ducts, the prevalence of which is 50/100 000 people. Common bile duct stenosis is a secondary outcome of chronic pancreatitis which occurs in 8-30% of patients. The obstruction of biliary duct is often associated with liver cirrhosis and previous studies have shown that the fibrosis of liver and pancreas can be diminished or the organs can regenerate when the ducts are opened (see references below). While I do not want to further discuss the disease itself, but rather focus on its treatment and prevention with use is metal-based stents (elaborated on below), here are two links to the websites which provide an overview of chronic pancreatitis and pancreatic fibrosis: https://pancreapedia.org/reviews/introduction-to-pancreatic-disease-chronic-pancreatitis, https://pancreapedia.org/reviews/pancreatic-fibrosis.

 

The treatment of stenosis of common bile duct (or pancreatic duct) is mainly done through drainage and opening of the ducts. This can be performed by surgical or endoscopic drainage and use of endoprosthesis such as stents. Several types of stents have been developed and used in the past, the most common types are plastic and metal stents. Metal stents often are covered and based on this are further subdivided into uncovered self-expandable metal stents (USEMS), partially covered self-expandable metal stents (PCSEMS), and fully covered self-expandable metal stents (FCSEMS).

Clinical trials generally agree that placement of PCSEMS and FCSEMS as opposed to USEMS and plastic stents results in better clinical outcomes. The commercially available metal stents are made of Nitinol (Ni-Ti alloy), Platinol (Pt-Ni-Ti), Elgiloy (Co-Cr-Ni alloy), or Stainless steel and their structures vary depending on whether they’re braided or laser-cut. The different alloys differ in terms of their expandability in the duct, elasticity, and thus ability to adjust to the shape of the bile duct. In general, Nitinol- based SEMS perform better as they are more elastic. Moreover, the patency of such stent was shown to be better in multiple studies, and the axial force (explained below) is generally lower than for steel- or Elgiloy-based stents which leads to lower dislocation rate of the Nitinol-based stent. Regardless of the materials from which they are made, the major issue with USEMS is that the bare metal directly interacts with the tissue, so hyperplasia and ingrowth of tissue between the mesh is often observed. Such a stent is then difficult to remove and very often fails as the overgrowth of cells again occlude the duct at the site of stent placement. Therefore, surgeons prefer to use PCSEMS and FCSEMS as opposed to USEMS.

The stents can be covered with various biomaterials, including polyurethane, silicone, or polytetrafluoroethylene. Those materials are relatively resistant to hydrolysis and therefore the coatings are durable in the duct. It has been suggested that covering of metal stent with membrane of approximately 50 um was enough to minimize hyperplasia and ingrowth of tissue but maintain the biodegradable properties of the membranes. This results in the increase of patency of the stent and allows for its removal when the stent is no longer needed (figure on the left presents examples of PCEMS and FCSEMS). The drawback of such design is that the stents could potentially migrate and therefore anchoring fins, flaps, or have dilated, flared ends are included in the design which reduce dislocation of these stents. The definite benefit of the covering is that the stents could include drug-releasing systems with for example anti-inflammatory agents or other drugs beneficial in treatment of pancreatitis.

As for the mechanical properties of stents, two major values are the most important in defining the stent’s characteristics, the axial force (AF) and radial force (RF). AF is the force applied onto a bend of a stent which results in straightening of the stent, and RF is the force applied by the stent onto the surrounding tissue by the expansion of the stent. Ideally, the stents should have low AF (no occlusion) and medium RF (medium force exerted on the duct itself, full expansion of the stent). The tables below summarizes some of the commercially available stents, their coverings, as well as AF and RF, migrations rate, and structure. SEMS come in different sizes, however studies have shown that regardless of their diameter, the clinical outcomes are very similar.

It seems that SEMS with coverings are very tunable in terms of their mechanical properties (), durability, antimigratory design without causing hyperplasia and maintaining the ease of removal of the stent, and can be further modified to include drug-releasing coatings and improve the overall efficiency of the stents as treatments for biliary duct/ pancreatic duct occlusion, and ultimately prevent chronic pancreatitis and formation of fibrotic scarring of the pancreas and liver.

References:

Behm, B., Brock, A., Clarke, B. W., Ellen, K., Northup, P. G., Dumonceau, J. M., & Kahaleh, M. (2009). Partially covered self-expandable metallic stents for benign biliary strictures due to chronic pancreatitis. Endoscopy, 41(06), 547-551.

Hammel, P., Couvelard, A., O’toole, D., Ratouis, A., Sauvanet, A., Fléjou, J.F., Degott, C., Belghiti, J., Bernades, P., Valla, D. & Ruszniewski, P., 2001. Regression of liver fibrosis after biliary drainage in patients with chronic pancreatitis and stenosis of the common bile duct. New England Journal of Medicine, 344(6), pp.418-423.

Isayama, H., Nakai, Y., Kawakubo, K., Kogure, H., Togawa, O., Hamada, T., Ito, Y., Sasaki, T., Yamamoto, N., Sasahira, N. & Hirano, K., 2011. Covered metallic stenting for malignant distal biliary obstruction: clinical results according to stent type. Journal of Hepato‐Biliary‐Pancreatic Sciences, 18(5), pp.673-677.

Isayama, H., Nakai, Y., Toyokawa, Y., Togawa, O., Gon, C., Ito, Y., Yashima, Y., Yagioka, H., Kogure, H., Sasaki, T. & Arizumi, T., 2009. Measurement of radial and axial forces of biliary self-expandable metallic stents. Gastrointestinal Endoscopy, 70(1), pp.37-44.

Shamah, S., Waxman, I., Chapman, C. G., Haider, H., & Siddiqui, U. D. (2018). Sa1293 Largers US experience with partially covered self expandable stents (SEMS) for malignant biliary obstruction: size does not matter. Gastrointestinal Endoscopy, 87(6), AB200.

Bulk Metallic Glasses (BMGs) are a newly emerged class of biomaterials that possess a unique combination of properties. BMGs thanks to their metallic component resemble metal-based biomaterials in their strength and stiffness, however as amorphous structures which components and internal organization can be easily altered they are also highly elastic (compressive elasticity), ductile, wear-resistant and generally do not cause a severe immune reaction. Moreover, they are easily modifiable both in terms of their macroscopic and microscopic structure (for example of surface molding patterns see figure below), which might positively influence their biocompatibility and biointegration by decreasing the formation of fibrous capsule around the BMG-implant.

While there are many types of BMGs, containing several metals, such as Zr, Cu, Fe, Ti, or V, among others, after reviewing some literature on the topic I found that most of now used BMG-based implants result in eventual isolation of the implant from host tissue by fibrous encapsulation (see figure on the left; examples: Mg₆₀Zn₃₅Ca₅ in porcine abdomen, Zr₆₀Ti₆Cu₁₉Fe₅Al₁₀ alloy in rabbit). However, the group of Kyriakides, research of which we learned about during the guest lecture, looks into how altering the surface of BMGs can improve their incorporation into the host tissue, that is result in lower inflammatory response and ultimately absence of fibrous encapsulation. The groups produced BMGs with different size nanopatterns on their surface by thermoplastic forming (to avoid the crystallization) and  investigated the response of macrophages and fibroblasts to the nanopatterned BMGs as these are the main cell types involved in inflammatory response, and production of collagen I-rich matrix and encapsulation respectively, and result in loss of functionality of implant.

Interestingly, the nanopatterns on the surface of BMGs resulted in lower fused macrophage – giant cell response, as well as smaller size of fibroblasts on the highly nanoscale-patterned material. The smaller size of fibroblasts can be explained by decreasing adhesion of the cells when the pattern was smaller which was also observed in their study. What is more important, these fibroblasts also expressed less collagen I, which is a very promising result and indication for future projects involving production of highly biointegrative biomaterials.

Based on these results, nanopatterning of BMGs seems to facilitate alteration of cellular responses to implants and therefore in the future we may be able to find the optimal nanopattern to fully diminish fibrous encapsulation implants, allowing for the ideal situation where integration of the material into host tissue would occur.

Silicone implants have been widely used in medical applications, as orthopedic or breast implants, in catheters and drains, to name a few, however, the problem with silicone is that it causes excessive extracellular matrix formation around the implant, possibly because it does not allow for cell attachment and thus incorporation into the tissue. The fibrotic capsule that forms often has a higher Young’s modulus that the soft tissue and therefore is more stiff than the surrounding tissue (in the case of silicone breast implants for example the adipose tissue).

To address the problem of high inflammatory response resulting in fibrosis of tissue surrounding silicone implants, the group from ETH Zurich investigated the use of Bioglass 45S5® in combination with silicone to optimize the bioinert properties of implants and improve its integration into the tissue. They produced silicone composites containing micro- and nano-particles of Bioglass (45 wt% SiO2, 24.5 wt% Na2O, 24.5 wt% CaO, 6 wt% P2O5). Additionally, their particles were porous which allowed for increased “exposure” of tissue to the incorporated Bioglass, which the group actually showed when they compared adhesion and integration into the tissue of the different composites. It is also worth mentioning that the group even observed less fibrosis and even some vascularization in the close proximity to the implant!

As silicone is often used in bone graft implants, they found that incorporation of Bioglass allowed for transformation into hydroxyapatite on the surface of the composites. This makes the bioglass-silicone composites more biocompatible and suitable for use in bone implants.

Interestingly, the mechanical properties of these composites proved to be influences by the size of incorporated Bioglass particles. In class we learnt that decrease in size of glass and ceramic particles increases their strength, and affects their elastic modulus. The paper shows that incorporation of microparticles results in lower modulus of elasticity than for nanoparticles, when both are implanted into biological system (in this case their studies were done in ovo). This means that by incorporating bioglass into silicone implants we can increase their integration into tissue (the paper actually showed that Bioglass-silicone composites are more integrated into the tissue than silicone-only composites), and decrease the formation of fibrous capsule.

Their results seem to show that incorporation of Bioglass can help optimize the bioinert properties of silicone-based implants so that they allow for cell attachment, and improve integration into the tissue without causing chronic inflammation, and eventually inducing fibrosis.

Article:

Cohrs, N. H., Schulz‐Schönhagen, K., Mohn, D., Wolint, P., Meier Bürgisser, G., Stark, W. J., & Buschmann, J. (2018). Modification of silicone elastomers with Bioglass 45S5® increases in ovo tissue biointegration. Journal of Biomedical Materials Research Part B: Applied Biomaterials.

This week in class we briefly touched upon the body’s immune reaction to the foreign body implantation, and how this reaction is dependent on the characteristics of the material and the extent of injury. While looking for adequate literature in relation to fibrosis, I decided to elaborate on how different types of materials induce more or less severe inflammation which later on results in formation of fibrotic capsule around the implant, using a simple example from a recent publication by Bertrans et al. (2018).

Biomaterials used as implants should be designed in a way that allows for mitigation of the inflammatory response and their integration into the tissue. Otherwise, biomaterial implants frequently cause the so-called foreign body reaction, which comprises of 1) protein adsorption and formation of matrix around the implant, 2) acute phase inflammation with presence of mast cells and granulocytes, this can further develop into 3) chronic inflammation where macrophages play a central role, 4) granuloma with presence of giant cells, and finally, through the activation of fibroblasts, 5) formation of the fibrous tissue surrounding the implant and isolating it from the peri-implant cells.

The study of Bertrans et al. followed-up on patients with hip replacements made out of and coated with different materials (ceramic on ceramic – CoC, ceramic on polyethylene – CoP, and metal on metal – MoM), who required revision surgery. The ceramic materials used for these implants were alumina-toughened zirconia (nanosized alumina in zirconia) – ATZ, and zirconia-toughened alumina (nanosized zirconia grains in alumina) – ZTA.

Interestingly, they found that in the patients with CoC prosthetics, the peri-implant environment consisted of much denser fibrotic capsule than in patients with MoM or CoP implants, and that increase was elevated with time after implantation of artificial hip joint (see the graph below). They were also able to find worn CoC in the connective tissue matrix.

The group further investigated the mechanisms underlying the differences between cellular reactions to these materials in vitro, and performed studies on fibroblast cultures, where the cells were cultured on surfaces identical to the ones of CoC, CoP, and MoM prosthetics. As a measure of the fibroblast response they looked at the levels COX2, a marker of chronic inflammation, and found an increase of that marker only in fully ceramic implants (especially the ATZ), suggesting increase of fibroblast response in the inflammatory reaction. They also conducted studies on peripheral blood mononuclear cell cultures and saw an increase of pro-inflammatory cytokines (IL-1, IL-6, COX2) when in contact with ATZ, but not with the other materials.

Their results clearly show that the reaction of the body to the foreign material can vary depending on the chemical and physical properties of the biomaterial used as an implant. In this specific case, the increase in inflammatory reaction and, as a consequence, fibrosis could be due to the presence of the worn ceramic particles in the tissue surrounding implant which might be bioactive, even though the wear of ceramics in general is rather low. While in the case of joint replacement, extensive fibrosis might prove to be beneficial and reduce the implant dislocation, for instance in the case of heart valve replacements extensive inflammation and fibrosis can be lethal.

A way to reduce this effect in biomaterial implants or drug delivery systems is for example by coating the surface of materials with protein which would mediate the reaction to the material.

Examples of biomaterials coated with protein and ways of their production are described for instance in the US Patent Application # US20120114734A1. The authors of this invention also suggest using compositions with covalently attached anti-inflammatory compounds to decrease the foreign body reaction. To me, it seems the most promising now, and easy-to-apply into clinical use way to diminish tissue fibrosis following implantation.

 

References:

Bertrand, J., Delfosse, D., Mai, V., Awiszus, F., Harnisch, K., & Lohmann, C. H. (2018). Ceramic prosthesis surfaces induce an inflammatory cell response and fibrotic tissue changes. Bone Joint J, 100(7), 882-890.

Scars. We all have them. Whether they come from a time when we fell on a bike while rushing on a bumpy road or remind us of a surgery that saved our lives when we had appendicitis, they are clearly distinguishable from other surfaces of our skin. In an ideal situation wound healing and tissue regeneration would occur after a skin injury, and the scar formation would be minimal. However, this is often not the case and many cuts, burns, etc. result in visible scar formation. This wound healing is dependent on a very careful interplay between several cell types, the cytokines, factors, proteins that they release, and slight changes might result in different healing outcomes which we see as more or less severe scarring.

Silver has been used in  medical treatments of wounds for centuries already, as it was known for its antimicrobial activity but only since recently researchers have put more attention into the use of silver in inducing successful wound healing and prevention of formation of the so-called hyperthrophic scars.

Silver nanoparticles (AgNPs) are a perfect example of inorganic nanoparticles, which are nowadays becoming increasingly used in medical applications. AgNPs specifically can be produced in different ways, mainly through physical, (bio)chemical, and biological approaches, and their exact physicochemical structure depends on the way of their production (they can for example be coated with silica).

Their sizes vary from study to study but most AgNPs are of about 20 nm in size, which seems rather small as nanoparticles can have up to 100 nm.

The advantage of using these nanoparticles is that they can penetrate the cell membrane, and their large surface area to volume ratio allows for more interaction site, and provoke a quicker reaction. Interestingly, studies have shown that topical treatment with AgNPs results in faster wound healing. Even though the exact mechanisms of AgNPs actions that would mediate the healing and skin regeneration remain a question, studies have found that application of AgNPs to wounds mediates inflammatory cytokine expression. Particularly, the expression of INF-gamma, TGFbeta-1, and IL-6 are altered, and as a result the inflammation at the site of injury is limited with AgNPs are applied. This could possibly explain why AgNP-treated wounds heal with less scaring. Several interesting studies have also been able to show that applying silver to wounds is more beneficial to skin tissue regeneration when the silver is administered topically as AgNPs in solution, and not in other forms (in this case in a silver sulfadiazine cream), and seem to allow almost full recovery and re-epithelialization without scar formation.

 

Additionally, studies on human fibroblasts, a cell type especially important in the deposition of collagen-rich matrix that forms scars, have shown that upon treatment with AgNPs the fibroblast growth is largely inhibited. All of these exciting results further speak for the idea of using nanoparticles as drugs or in drug delivery which we recently discussed in class. It seems like AgNPs offer a promising means of mediating proper wound healing and diminishing scar formation which could be used in treatment of skin lesions and burns. Hopefully soon we will be able to use them!

Scar formation occurs in tissues as a reaction to chronic inflammation or severe injury, where the scar formed in the body’s attempt to repair the damaged site. It most often results in severe functional impairment of the affected organ. The regeneration of healthy tissue at those sites is largely limited because of the presence of abnormal extracellular matrix and absence of blood vessels.

I am particularly interested in, and will try to focus my blog posts on exploring the novel biomaterials used in facilitating the regeneration of these fibrotic tissues, preventing the formation of scars, as well as finding why and how some biomaterials are the cause of tissue scarring. I am hoping to cover a wide range of topics, and explore applications in different types of tissues, such as heart, lung, brain, or liver. Enjoy!