1

Synthesis and Characterization of Extracellular Matrix

Mimetics New synthetic glycol-tools for cell cultures

Inês Figueiredo

inesahfigueiredo@tecnico.ulisboa.pt

Instituto Superior Técnico, Universidade de Lisboa, Portugal March 2018

Abstract

Tissue engineering relies on the possibility to engineer cell microenvironments by means of

bioactive materials, biochemical and physical stimuli in order to guide cell behavior and to regenerate damaged tissue by pathologies or trauma. Extracellular matrix inspired scaffolds are important tools useful to mimic and control cell microenvironment, to guide and stimulate cell processes involved in tissue regeneration. The aim of this work is the synthesis of new glycosylated matrix mimetics that can be used for different applications such tissue engineering and cell biology studies in physiologic and pathologic conditions. In this context, the development of new conjugation strategies to functionalize collagen and elastin scaffold with glycans was done. Carbohydrates, despite of being complex polysaccharides, they are relevant components of the cell environment and cell membrane, contributing to cell interactions at different levels. It is evident that the cellular microenvironment contributes to the spatial and temporally complex signaling domain that directs the fate of the cells and the idea is to mimic in vitro the same microenvironment that can be found in vivo. For that, a synthetic approach was created using three different approaches using chemoselective processes. In form of 2D matrices, the biomaterials, were bioactivated using carbohydrates and characterized by NMR analysis (1NMR), confocal microscopy, colorimetric/absorbance assays with ninhydrin and lectin analysis (by fluorescence and absorbance analysis). Keywords: tissue engineering, extracellular matrix, microenvironment, collagen, elastin

1. Introduction

Tissue engineering started to be developed and evolved through the development and use of biomaterials. This area is related to the combination of scaffolds, cells and biologically active molecules in functional tissues. When lesions exceed the human regeneration limit, therapeutic treatment is necessary. In this context, medicine is developing innovative approaches and clinical applications referred to as “regenerative medicine”. This medicine field is a broad area that includes tissue engineering but also incorporates research on self-healing – where the body uses its own systems, sometimes with help foreign biological material to recreate cells and rebuild tissues and organs. Regenerative medicine approaches often combine key essential components: signaling macro and small molecules (i.e., peptides, glycans, paracrine

factors that include growth factors, cytokines, interleukins, etc.), cells and biomaterials. [1] Both tissue engineering and medicine work together to improve tissue. Cells are considered as the building blocks of tissues and tissues the basic unit of function in the body. [2] In nature, cells gain a variety of information both from surrounding cells and from their microenvironments, that is, from the extracellular matrix (ECM). The ECM has a structural and biochemical role that is involved in several cell processes like cell adhesion, migration, proliferation and differentiation and they are natural biomaterials that can be bio-activated with signaling molecules. It is composed by three major classes of biomolecules, glycosaminoglycans (GAGs), fibrous proteins such as collagen, elastin and glycoproteins such as

2

fibronectin and laminin. The ECM in addition of being critical for connecting cells together to form the tissues, is also a substrate upon which cell migration is guided during the process of embryonic development and importantly, during wound healing and is responsible for the relay of environmental signals to the surfaces of individual cells. Finally, the ECM should be considered as a dynamic material, where the local medium can be remodeled through cell-mediated secretion and deposition of molecules or degraded through cell secreted enzymes called matrix metalloproteinases (MMPs). [3] The ECM microenvironment can be mimicked by extrapolation of key properties into biomaterial design. This type of environment is increasly recognized to have regulatory role for cell populations in pathological and healthy states. In this way, there are several material properties that can be adapted in order to influence the cell behavior. They embrace different physical parameters, such as morphology (i.e., fibers, sponges), roughness, topology and topography (at the micro and nano scale) and mechanical properties (i.e., stiffness). A good explanation of these effects is that mechanosensing is an active cellular process involving dynamic interplay between cells and their physical environment. Indeed, several studies demonstrate that physical signals potently guide cell fate and functions. Surface chemistry (i.e., exposed functional groups) is an extra relevant factor in the interaction with cells, once it influences wettability, protein interactions and cell behavior. The ECM is a very high dynamic environment that is locally degraded and remodeled during development, homeostasis, wound repair, and even pathophysiological events. [4] The degradation of the ECM firstly occurs through enzymatic cleavage by cell-secreted proteases, allowing cells to remodel their local microenvironment or moves through this dense matrix. To explore how stiffness and structure affect cell function, it is possible to create photodegradable hydrogels that enable material properties in space and time for the user-tunable. [5]

Figure 1: The extracellular matrix. [11]

1.1. Biomaterials Collagen is one of the naturally-derived polymers and is the most abundant protein in the ECM, which forms trimeric protein rods that provide tensile strength to the network. [5] The various collagens constitute the major proteins comprising in the ECM. There are at least 44 different collagen genes dispersed through the human genome that combine to create different collagen fibrils. Despite of many types of collagen have been characterized, only a few types are used to produce collagen-based biomaterials. The most used is type I. Collagens, as much as the majority of secreted proteins, are synthesized in the rough endoplasmic reticulum (rough ER). This polymer like all secreted and processed precursor proteins, originate as longer precursor proteins called preprocollagens. After removal of the signal peptide from the preprocollagen precursor, that occurs in the lumen of the rough ER, the remaining protein is referred to as a procollagen (or tropocollagen). This polymer is predominantly synthesized by fibroblasts, but epithelial cells are also responsible for the synthesis of some of the ECM collagen. The use of collagen-based biomaterials has a wide range of applications in vivo or in vitro. [3] Elastin is an important extracellular matrix protein that require elasticity. It is most abundant in organs where elasticity is of major importance, such as arteries (50%), lungs (30%), bladder, skin (2-4%), elastic ligaments, in geral, and cartilage and allows these tissues in the body to resume their shape after stretching or contracting. [6] The most important property of elastin is to confer flexibility and elasticity indispensable to the function of these tissues. [7] It is composed of soluble tropoelastin protein that contain primarily, glycine and valine, modified alanine and proline residues and another important property of the precursor of elastin, tropoelastin and elastin-like peptides is their potential to self-assemble under physiological conditions. Elastin can be used as biomaterial in various forms, including insoluble elastin in autographs, allografts, xenografts, decellularized ECM or purified elastin prepared first. [8] Elastin like Collagen is a protein found in connective tissues, but it is a different type of protein than collagen.

Figure 2: Structure of Collagen and Elastin. [12]

3

1.2. Glycans Carbohydrates are the most abundant biomolecules in nature and essential elements in a wide range of processes in living systems. One approach using carbohydrates is the use of epitopes because they can carry much more information per unit weight than do either nucleic acids and proteins. Surface carbohydrates on a cell can be serve as a point of attachment for other cells or infectious bacteria, viruses, etc. In this way, they also have an important role in determining the fate of cells because they mediate the migration of the cells during embryo development. The compounds that have proteins that are linked in a chemical way to the carbohydrates are called glycoproteins. Glyconanomaterials, nanomaterials carrying surface carbohydrates have merged and demonstrated increasing potential in biomedical imaging, therapeutics and diagnostics. They have an important role in cell fate regulation and in physiological and pathological methods (cancer, aging and other diseases). The sugar that we used were lactose and maltose in order to test the effect of last unit (galactose for lactose and glucose for maltose). [9]

Figure 3: Structure of carbohydrates. [13]

2. Approach The aim of my thesis work is the synthesis of new glycosylated matrix mimetics so, the idea is using synthetic ECM matrices that were prepared by functionalizing ECM-derived proteins (collagen and/or elastin) by: i) thiol-ene, ii) methoxamine iii) phenylhydrazine reactions creating the same in vitro microenvironment that can be found in vivo.

2.1. Collagen formulation

To study and design insoluble collagen, it was produced collagen films by a solvent-casting method. It was prepared two-dimensional (2D) scaffolds by a solvent casting method and the collagen matrices were produced as thin transparent films using collagen type I (from bovine Achiles tendon – Sigma-Aldrich CAS nº C9879). It was weighted 690mg of collagen and added in 315mL (0.5AcAH with 28,6 mL of Acetic Acid and 1L of water) and left for four hours under agitation and at 40ºC (to have a better solution the flask was cover with alluminium), then with a mixer occured the homogenation and finally with a sering with 20mL the solution was put on the plates in portions. The solution is left for one day under wood to dry and as soon as the collagen was dry, it was washed with MQH2O and one last wash with ethanol.

2.2. Materials and procedure

First, it was used material functionalization with glycidic structure via thiol-ene reaction with soluble collagen that consisted using thiol groups introduced on collagen matrices to allow a thiol-ene reaction between thiolated collagen and the sugar. 5,5'- Dithiobis (2-nitrobenzoic acid), DTNB, is a reagent able to form disulfide bridges with free thiol groups, and this reagent was used as a test to label and quantify inserted -SH groups.

Figure 4: Scheme of via thiol-ene reaction.

4

This method was tried with the biomaterials collagen and elastin. The elastin that was used during the work was soluble from bovine neck ligament. (CAS nº: 9007-58-3). It is salt-free (lyophilized powder). For the calculations it was used as reference for collagen and elastin:

4,5gofLysine----→100gofmolecule(1)

Each step of reaction was tried in different quantities, but the final quantities chosen to start were 50mg of collagen. The only reactions that was done for tests was the first step where the g - thioburyrolactone reacts with the collagen and that was done with an equivalent of reagent of 40 and with EtOH:PBS as a solvent. This reaction was considered fast, so it was only needed two hours under agitation. The other reaction that was done was the DTNB test were the equivalent of reagent also used was 40 and the solvent carbonate buffer. For the calculations, it was taken in consider the molecular weight, the quantities in mg and mmol, the equivalent of reagent, the volume and the density and for that the following equations were used:

𝑛 = 𝑚𝑜𝑓𝑙𝑦𝑠𝑖𝑛𝑒

𝑀𝑀𝑜𝑓𝐶𝑜𝑙𝑙𝑎𝑔𝑒𝑛(2)

𝑒𝑞𝑢𝑖𝑣𝑎𝑙𝑒𝑛𝑡 × 𝑚𝑚𝑜𝑙𝑜𝑓𝐿𝑦𝑠𝑖𝑛𝑒= 𝑚𝑚𝑜𝑙𝑜𝑓𝑡ℎ𝑖𝑜𝑏𝑢𝑟𝑦𝑟𝑜𝑙𝑎𝑐𝑡𝑜𝑛𝑒(3)

𝑚 = 𝑛 × 𝑀𝑀(4)

𝜌 = 𝑚𝑣(=)𝑣 =

𝑚𝜌

(5)

For solubility of collagen was used as reference:

10𝑚𝑔𝑜𝑓𝑐𝑜𝑙𝑙𝑎𝑔𝑒𝑛 − −−→ 1𝑚𝐿𝑜𝑓𝐻U𝑂(6)

The soluble collagen takes some tame to be completely dissolved so it was used as resource the vortex and a sonicator to help. Using as biomaterial, the elastin, the same procedure was used, and the start quantities were 10mg of elastin. It was also done only the first step with the g - thioburyrolactone and the DTNB test. The conditions used were the same as used with the biomaterial collagen. For the calculations the solubility of elastin used as reference:

100𝑚𝑔𝑜𝑓𝐸𝑙𝑎𝑠𝑡𝑖𝑛 − −−→ 1𝑚𝐿𝑜𝑓𝐻U𝑂(7)

Since it was difficult to choose the best wash to obtain the maximum product as possible, this approach wasn’t finished. It was tried three ways of wash: with centrifugation where it was used acetone to separate the product (3 x times); it was used the vivaspin flask and finally the dialysis procedure. The best one was the last one, dialysis but since it takes four days to do the complete wash, this approach wasn’t follow.to do the dialysis it was used a piece of cellulose membrane that was previous washed with MQH2O. The membrane was cut and open and with a syringe the product was put inside and after that closed. The membrane was left for four hours under agitation in a big gobble with MQH2O. The water on the first day needs to be change after every hour and in the next three days just one time. After that the product is removed from the membrane and put in a flask to be insert in nitrogen liquid and finally in a freeze dry for two days.

Then it was tried a second approach using material functionalization with glycidic structures via chloroacetone reaction in two ways:

I. With phenylhydrazine in 2ºStep, because with the phenyl group it was easier to analyze in NMR, so it is an advantage of using this method.

II. With methoxyamine, because it has a small group that won’t affect the attachment of the sugar.

This method was tried with the biomaterial insoluble and soluble collagen. First it was tried with methoxyamine in the second step. It was used soluble collagen but like in the other approach the best way to obtain the maximum product was using the dialysis procedure. The first step it was to obtain the ketone using as reagent chloroacetone. The solvent used was PBS with a pH=7,5, the equivalent of reagent used was 40 and the quantities of start material was 100mg. For the calculations the equations used was the same as used in the previous approach. For the second step it was used as reagent the methoxyamine. The solvent used was PBS with a pH=8 with an equivalent of reagent of 40 and it was used 50mg of the product obtain in the first step. On the third step it was used as reagent NaBH3CN with Citrate Buffer with pH=5 as solvent and 25mg of the product obtain in the previous step.

5

Figure 5: Scheme of via chloroacetone reaction in two different ways. The equivalent of reagent was also 40. Finally, in the last step it was used as reagent the sugar maltose and 10mg of the product obtain in the previous step. The solvent used was also citrate buffer with pH=5 and an equivalent of reagent of 40. All the products obtain in each step were washed with the dialysis procedure. Using as biomaterial the insoluble collagen, it was easier to wash. With this biomaterial was tried with the metoxyamine and the phenylhydrazine. The procedure was simpler because it wasn’t necessary to take in consider the solubility of the collagen. It was used in the first step with chloroacetone to improve the functionalization an equivalent of reagent of 100 with 10mg of start material and with PBS pH=7,5 (5mL) as solvent. On the second step with methoxyamine it was also used 100 as equivalent of reagent and all the product obtain in the first step was used in the reaction (10mg) with PBS with pH=8 (5mL) as solvent. On the third step all the product was used with NaBH3CN as reagent, 100 of equivalent of reagent and citrate buffer with pH=7,5 as solvent (5mL) and finally with maltose in the last step was also used 100 as equivalent of reagent and citrate buffer with pH=7,5 as solvent (5mL). With phenylhydrazine as reagent in the second step the conditions used were the same. All the steps had the same procedure as using methoxyamine with the only difference that the phenylhydrazine was a liquid and the methoxyamine a solid. The quantities used was the same (10mg of product in each step) with 100 of equivalent of reagent and the solvent used with phenylhydrazine was also PBS buffer with pH=8 (5mL).

To have a comparation between the results it was used the results that were tried before using material functionalization with glycidic structures via reductive amination with insoluble collagen. That was done by my professor Dr. Laura Russo. In this last method, the purpose was to study the migration of the cells and the maintain of them. This via, has the goal to be studied in cells of patients with lung cancer. These patients have stem cells that are resisting to chemotherapy, so the purpose was to characterize the effect of glycoenvironment of this process. The main purpose is to achieve the reductive amination by reacting free lysine side-chain amino groups of the collagen with sugars in the presence of a reducing agent. In the first reaction, the carbohydrate used was lactose that will be transformed in galactose and in the second reaction, the carbohydrate used was maltose that was transformed in glucose and the pH chosen was 6. The reductive amination, in all cases, has been performed in aqueous solution (citrate buffer pH 6.00) in the presence of the reducing agent NaCNBH3, producing a covalent stable neoglycosylation.

6

Figure 6: Scheme of via reductive reaction.

3. Characterization and discussion For characterized the products obtain it was done NMR analysis (1NMR), confocal microscopy, colorimetric/absorbance assays with ninhydrin and lectin analysis (by fluorescence and absorbance analysis). 3.1. 1NMR The aim of using this technique, is to see if the groups that were used in all the steps were connect to the lysine group of the collagen. The solutions for the reading were prepared by homogenization with Deuterium Oxide (CAS nº: 7789-20-0). The use of this component gives to the solution different nuclear properties that makes the reading for NMR easier. When the solutions had the product with insoluble collagen was also added Sodium Deuteroxide (CAS nº: 14014-06-3) to help in the dissolution of the collagen. As soon as the sample was denatured large increases in intensities in the upfield region some of which can be related to amino acids that are present in large amounts in collagen. In the first approach, via thiol-ene reaction, it is possible so see the change that happen in the NMR, specially between the signals at 8.9 and 7.5 ppm that are referred to the aromatic ring. Since we were able to introduce the ketone in one step this strategy wasn’t follow. This strategy can be used for subsequent chemoselective reaction to link small molecules in any cases.

Subtitle: Red – Collagen Control and Blue – 3rd step (DTNB)

Figure 7: Results of 1H NMR of product via thiol-ene reaction.

In the second approach, via chloroacetone with soluble collagen as biomaterial, with the spectrum using this method, it was easier to see the changes that happened in the conformation in 2.4 and 2.8 ppm and 1.3 and 1.6 ppm. The lysine methylene signal that increases at approximately 3.00 ppm, indicates the modification of collagen.

NH2

Collagen

HN

Collagen S-TNB

O

S S NO2

O2N

O2N

7

Subtitle: Red – Collagen Control, Green – 1st step, Dark Green – 2nd step, Blue – 3rd step and Purple – 4th Step

Figure 8: Results of 1H NMR of product via chloroacetone reaction in all steps

Using insoluble collagen with methoxyamine in the second step it was obtained results with more quantities of reagents. When the sample was denatured large increases in intensities in the upfield region some of which can be related to amino acids that are present in large amounts in collagen. These are: glycine Hd 3.8 to 4.0 ppm; proline Hd, 3.5 to 3.7 ppm; proline Hd, 1.9 to 2.3 ppm; hydroxyproline Hd, 3.7 to 3.9 ppm; hydroxyproline HO, 2.1 to 2.3 ppm; alanine methyl Hd, 1.3 to 1.4 ppm; valine, leucine, and isoleucine methyl Hd, 0.8 to 1.0 ppm. Also, here it is possible to see that the lysine methylene signal at approximately 3.00 ppm, indicates the modification of collagen. This spectrum corresponds to the master-piece and the final step with the sugar attached. Subtitle: Blue – Collagen Control and Red – 4th Step using methoxyamine in 2nd Step

Figure 9: Results of 1H NMR of product via chloroacetone reaction comparing the masterpiece and the last step using methoxide.

Using insoluble collagen but with penylhydrazine in the second step, it is easy to see that the conformation changed a lot, special between 2.5 to

4.5 ppm, here specially at 3.0 ppm we have the signal that change a lot. It is easy to conclude that almost all the conformation suffers a change, so this method was the chosen to be the most successive. In here, the spectrum also corresponds to the master-piece and the final step with the sugar attached. Subtitle: Blue – Collagen Control and Red – 4th Step using phenylhydrazine in 2nd Step

Figure 10: Results of 1H NMR of product via chloroacetone reaction comparing the masterpiece and the last step using phenylhydrazine.

3.2. Fourier transform infrared (FT-IR) spectroscopy The aim is to see the different of absorbance between the reactions. It was possible to evaluate if the chemical process had any damaging effect on supramolecular structure of neoglycosylated collagens. This analyze was only done via chloroacetone with soluble collagen. The spectra show the analysis of functionalized and not functionalized collagen samples. It is possible to see the increase of absorbance when compared the first step (green line) with the last step (purple line) and with this raising in the analyses of the external layers of the collagen matrix the neoglycosylation reaction happened successfully. Due to the functionalization of the collagen samples be on surfaces was performed a plot (panel B) analysis in glycan typical spectra zone (1200-1000 cm-1 region), in which it is possible to see the increase of value for the glycosylated sample, as proof of confirmation of the functionalization with sugar.

1.01.52.02.53.03.54.04.55.05.56.06.57.0f1(ppm)

1

2

Collagen

2

IFN-2-65_2

1

0.00.51.01.52.02.53.03.54.04.55.05.56.06.57.07.5f1(ppm)

1

2

Collagen

2

IFN-2-64

1

NH

O

NH2

Collagen

NH

NO

O

OHOHHO

HO O

OOHHO

HO

NH2

Collagen

NH2

Collagen

NH

N NH

O

OHOHHO

HO O

OOHHO

HO

NH

NO

O

OHOHHO

HO O

OOHHO

HO

NH

HN

O

NaBH3CNNH

N

Acetate BufferpH=5

O

NH

O

NH

HN

O

MeONH2 NaBH3CNNH

NO

O

OHOHHO

HO O

OOHHO

HO

CH3COCH2ClNH

NO

Maltose

Acetate Buffer pH=5PBS Buffer

pH=7,5PBS BufferpH=8

Acetate BufferpH=5

NH2

Collagen (Insoluble)

8

Subtitle: Blue – control, Green – 1st step, Dark Green – 2nd step, Purple – 3rd step, Red – 4th step

Figure 11: Results of FT-IR of product via chloroacetone reaction in all steps.

3.3. Colorimetric/Absorbance Assays 3.3.1. Ninhydrin tests The aim, of using ninhydrin is that this molecule has the particularity of link to free amino groups. In this way, if the solution stays colorless it means that the ninhydrin didn´t react with the lysine of the collagen and if the solution become purple, it means that the ninhydrin reacted with the lysine and the group that was supposed to link, didn’t link like was suppose, or if it linked, didn’t is in the best proportion. The Ninhydrin used became from Sigma-Aldrich. The reaction between the alfa-amino acid (lysine) and the ninhydrin involved is:

𝑎𝑙𝑝ℎ𝑎 − 𝐴𝑚𝑖𝑛𝑜𝐴𝑐𝑖𝑑 + 𝑁𝑖𝑛ℎ𝑦𝑑𝑟𝑖𝑛→ 𝑅𝑒𝑑𝑢𝑐𝑒𝑑𝑛𝑖𝑛ℎ𝑦𝑑𝑟𝑖𝑛+ 𝑎𝑙𝑝ℎ𝑎 − 𝑎𝑚𝑖𝑛𝑜𝑎𝑐𝑖𝑑+ 𝐻_2𝑂(8)

Step 1 – Oxidative Deamination reaction where two hydrogens from the alpha-Amino Acid are removed to yield an alpha-Amino Acid. At the same time, the original Ninhydrin is reduced and loses an oxygen atom with the formation of water.

𝑎𝑙𝑝ℎ𝑎 − 𝐴𝑚𝑖𝑛𝑜𝐴𝑐𝑖𝑑 + 𝐻_2𝑂→ 𝑎𝑙𝑝ℎ − 𝐾𝑒𝑡𝑜𝐴𝑐𝑖𝑑+ 𝑁𝐻_3(9)

Step 2 – The NH group in the alpha-Amino Acid is hydrolyzed and form an alpha-Keto Acid with the formation of Ammonia.

𝑎𝑙𝑝ℎ𝑎 − 𝐾𝑒𝑡𝑜𝐴𝑐𝑖𝑑 + 𝑁𝐻d → 𝐴𝑙𝑑𝑒ℎ𝑦𝑑𝑒 + 𝐶𝑂U(10)

Step 3 – Under heat conditions, the alpha-keto

undergoes decarboxylation reaction to form an aldehyde with less carbon atom than the original Amino Acid.

𝑎𝑙𝑝ℎ𝑎 − 𝐴𝑚𝑖𝑛𝑜𝐴𝑐𝑖𝑑 + 2𝑁𝑖𝑛ℎ𝑦𝑑𝑟𝑖𝑛 → 𝐶𝑂U +𝐴𝑙𝑑𝑒ℎ𝑦𝑑𝑒 + 𝐹𝑖𝑛𝑎𝑙𝐶𝑜𝑚𝑝𝑙𝑒𝑥𝑃𝑢𝑟𝑝𝑙𝑒 +3𝐻U𝑂(11) [10]

Figure 12: Comparation between the control solution (purple solution) and the solution with the product (colorless solution).

With the analyses of the images, it was proved that, since the reaction stayed colorless, the ninhydrin didn’t react with the lysine of the collagen, so the attachment occurred. The solution that is purple is the solution with the collagen as control. 3.3.2. 2,4 – dinitrophenylhydrazine Tests The aim is using a solution with 2,4 – dinitrophenylhydrazine, that is composed of drops of the component that we want to test with 2mL of ethanol. This test is selective for ketone groups, in this way, if the reaction occurs there is the precipitation of the product.

Figure 13: Comparation between the control solution (right flask) and the solution with the product (left flask).

With the analyses of the images, it was proved that, since didn’t occurred a precipitation of the solution, the reaction between the 2,4 – dinitrophenylhydrazine and the ketone group didn’t happen, so it was another proof that the functionalization occurred. The solution on right, it was about the collagen as control.

Carbohydrates

9

3.4. Biological Assays

3.4.1 Confocal Microscopy The aim of using confocal microscopy, is to see the glycan recognition, in other words the goal of using this technique is to evaluate the performance of the glycomaterial. In this methodology, was used lectins that reacted with glycosylated materials and with the collagen control, to test if the recognition occurred in the best way. By lectin interaction we analyzed not just the presence of glycans but also the correct exposition on material surface. The lectins frequently appear on the surface of cells, where they are strategically positioned to interact with the carbohydrates. So, the purpose is to perform the same environment but in vitro. Like the surface of the carbohydrates, the surface of the lectins goes through changes that coincide with the cell’s physiological and pathological states. [29] The lectins used were Concanavalin A from Canavala ensiformis (Jack bean) in lyophilized powder form. Lectins suspensions were added on glycosylated collagens and collagen control. After that, the solution was left under incubation for two hours. To do the washes, was used a solution with PBS (6 times per 15min each) and finally the samples were analyzed. W here the molecular form of each is:

Figure 14: Images obtain with Confocal microscopy. The first one represents the Collagen a), the second b) and the third c). The control doesn’t show fluorescence, and this is due to the lectin isn’t able to link sugar. On the other hand, in the glycosylated collagen (b and c right images) we can see the lectin recognition by FITC fluorescence. 3.4.2 MTT Assay The MTT assay is a colorimetric assay to assessing cell metabolic activity. It is used NAD(P)H, where dependent cellular oxidoreductase enzymes will, under defined conditions, reflect the number of viable cells present. This assay was done using as samples the products obtain in via chloroacetone, the collagen itself and

via reductive amination that was done before by my colleagues in the University of Milano-Bicocca. The assay was done by another colleague of the university. Subtitle:

Figure 15: Comparation of the results obtain for mitochondrial activity via chloroacetone, via reductive amination and the collagen itself.

3.4.3 LDH Assay The biocompatibility of collagen and glycan-modified collagen was assessed using human neuroblastoma cells (SH-SY5Y). Cells were grown on collagen for 48h and the cytotoxicity was evaluated by LDH and MTT assays. Results show that the presence of collagen does not affect cell viability of SH-SY5Y. A reduction of mitochondrial activity was observed only in cells grown on collagen 2 chloroacetone reaction). The assay was done by another colleague of the university.

Figure 16: Comparation of the results obtain for cell viability via chloroacetone, via reductive amination and the collagen itself.

HN

O

OH

OHHO

HOO

OHOH

HO

HO

1

NH

NO

O

OHOHHO

HO O

OOHHO

HO

NH

NO

HO

OOHHO

HO

2 3

NH2

Collagen

NH

NO

O

OHOHHO

HO O

OOHHO

HO

NH

NO

HO

OOHHO

HO

2 3

10

4. Final Conclusions In a specific tissue repair by tissue engineering, the natural Extracellular Matrix (ECM) of this tissue with hierarchical structure and mechanical behavior should be ideal scaffold. My thesis was conducted to develop functionalized by biomaterials based on soluble and insoluble collagen and soluble elastin. The development of collagen films and soluble collagen functionalized with different sugars like maltose and lactose were where analyze and characterized by three different strategies. Carbohydrates, due to their important role in biology, proved that they were good choices to do the functionalization of these biomaterials and it was possible to study their potential effect on different biological systems despite of there is a lot of work to do yet. The three different strategies for functionalization had in common the fact that free lysine side-chains amino groups have been useful for collagen modifications. All the products obtained were characterized in term of their functionalization by NMR and FTIR. Furthermore, in order to access if the exposed sugar can also exploit their biological signaling functions upon recognition of it is complementary receptor, biological assays, colorimetric/absorbance assays like ninhydrin and 2,4 – dinitrophenylhydrazine tests, Confocal Microscopy using lectins, MTT and LDH were done also. The results obtained were very interesting providing the relevant role of carbohydrates as signaling cues when covalently linked to biomaterial surfaces like collagen, as was demonstrated by the physio-chemical characterizations made. Finally, since it wasn’t possible to proceed with elastin, deeper studies are needed in order to try a different biomaterial from collagen. To clarify the role of different carbohydrates in the differentiation processes of different cell lines, can be uses in these studies another carbohydrate to have more information to compare.

5. References [1] Lee, E. J.; Kasper, F. K.; Mikos, A. G. Ann. Biomed. Eng. 2014, 42(2), 323–337 [2] Andrades, J. A. (2013). Regenerative Medicine and Tissue Engineering. InTech [3] Medical Biochemestry. Access in December 10 2017. Available on http://themedicalbiochemistrypage.org/extracellularmatrix.php. [4] Russo, L., Cipolla L. (2016). Glycomics: New Challenges and Opportunities in Regenerative Medicine. ChemInform. 47 (46). [5] Kyburz, K. A., Anseth K. S. (2015)., 43 (3), 449-500. [6] Karsdal M.A., Leeming D.J., Henriksen K., Bay-Jensen A.C., Biochemistry of Collagens, Laminins and Elastin, 30, 2016. [7] Glagov, S.; Vito, R.; Giddens, D. P.; Zarins, C. K. J. Hypertens. Suppl. 1992, 10(6), S101-104. [8] AnnMarie. Acess in January 13 2018. Available on https://www.annmariegianni.com/whats-difference-collagen-elastin/. [9] Wang X., Ramstrom O., Yan M., 2010, 22, 1946-1953. [10] Wang, Sun N., Amino Acid Assay bcy Ninhydrin Colorimetric Method, MD 20742-2111 [11] Access in December 15 2017. Available on https://ka-perseus-images.s3.amazonaws.com/2743d345dabf839cffe83a7474260f3c23730d91.png [12] Acess in January 13 2018. Available on https://www.annmariegianni.com/whats-difference-collagen-elastin/. [13] Russo L., Sgambato A., Guizzardi R., Lecchi M., Pastori V., Petecchia L., Gavazzo P., Vassalli M., Cipolla L., Nicotra F., Scaffolds for Neuronal Regeneration, 2016