FI20236062A1 - Controlled release nanocellulose compositions and methods for manufacturing thereof - Google Patents

Abstract

The disclosure relates to controlled release nanofibrillar cellulose compositions and methods for manufacturing thereof, and particularly to applications of nanofibrillar cellulose compositions in wound treatment.

Description

CONTROLLED RELEASE NANOCELLULOSE COMPOSITIONS AND METHODS

FOR MANUFACTURING THEREOF

FIELD OF THE DISCLOSURE

The disclosure relates to controlled release nanofibrillar cellulose compositions and methods for manufacturing thereof, and particularly to applications of nanofibrillar cellulose compositions in wound treatment.

BACKGROUND OF THE DISCLOSURE

The skin has a major role in providing protection for the body against environmental changes and controlling the overall thermal and moisture balance. After an injury caused by an external trauma reaching the epidermis or deeper tissue layers, the integrity of the skin is restored with complex, multistep wound healing including four overlapping phases: hemostasis, inflammation, proliferation, and remodeling. Various cell types, including keratinocytes, fibroblasts, endothelial cells, and immune cells, like macrophages, neutrophils, and lymphocytes, take part in wound healing enhancing the prevention of pathogens and inducing neo-epithelization, granulation tissue formation, and angiogenesis of the injured cutaneous site. The harmony of the complex wound healing process is highly controlled by growth factors, including cytokines and chemokines, which are released during paracrine signaling of the cells. These bioactive compounds participate in every wound healing step determining cell proliferation, migration, differentiation, and homeostasis in extracellular matrix production, which can be disturbed whether growth factor concentration is unbalanced.

Although wound healing is a self-repairing process, additional wound care is required to provide immediate coverage and a suitable environment as well as avoidance of infections or other complications that prolongs the healing. Especially in the case of extensive & 25 cutaneous wounds, tissue engineering has aimed to develop skin substitutes including

N cells and cell-released bioactive compounds that actively participate in tissue

S regeneration. These cell-based systems provide enhanced cell proliferation, and natural

N stimulation via paracrine signaling leading to enhanced wound healing. Utilization of these = products in wound healing applications has even led to a decrease in the number of chronic a 30 wounds associated deaths. However, culturing of autologous cells for tissue regeneration

S can be a time-consuming and expensive method, requiring controlled facilities for their 2 production, which is why improved solutions are reguired.

N Instead of only pursuing passive mechanical protection and bio-inertness, wound care development has aimed more at bioactive participation in wound healing to induce tissue regeneration at the site of action. Tissue regenerative approaches, like cell- and growth factor-based skin substitutes, have shown potential results in regeneration or even replacing injured cutaneous sites. However, due to a wide variety of wound types and different needs in every wound healing phase, requirements in wound treatment have not yet been fulfilled. Challenges in skin substitute development have been faced in the efficacy of the bioactive factors after transplantation, feasible and readily available sources for the wound products in addition to the possible adverse effect of the used scaffold material, like allergic reactions or host rejection due to the xenogeneic origin.

BRIEF DESCRIPTION OF THE DISCLOSURE

An object of the present disclosure is to provide nanofibrillar cellulose compositions, a nanofibrillar cellulose composition for use in wound treatment, use of the nanofibrillar cellulose composition for manufacturing a wound treatment product, a wound treatment product comprising the nanofibrillar cellulose composition, a method of producing a nanofibrillar cellulose composition, and a kit so as to overcome the above problems.

The object of the disclosure is achieved by the composition, product, use, method and kit which are characterized by what is stated in the independent claims. The preferred embodiments of the disclosure are disclosed in the dependent claims.

BRIEF DESCRIPTION OF THE DRAWINGS

In the following the disclosure will be described in greater detail by means of preferred embodiments with reference to the accompanying drawings, in which

Figure 1 shows scratch wounds right after creation (A) and after the administration of the formulations (B). In this image, the wound on the right was treated with T11 and left-sided with NaCl 0.9% as a control. Abbreviations: TI = Test item, NaCl = Sodium chloride; n Figure 2 shows visual observation and measurement of wound contraction. Treatment

N formulations (pointed with arrows) including NFC were self-detached from the wound site a 25 (A, B, pictures from the mice treated with TI1). In the case of TI3 in which NFC was

S degraded, no treatment formulations were observed (C). Wound contraction was i measured by comparing the size of the wound on day 9 to the starting point on day 0 (D,

E treatment groups n: 5-6, control n:18). Abbreviations: NFC = Nanofibrillated cellulose, TI

N = Test item; 2 30 Figure 3 illustrates macrophage staining indicating the presence of macrophages in total

O (A, C, E, G) and activated macrophages (B, D, F, H). Although TI3 showed a significantly higher intensity of Iba-1 stain (1), the presence of active macrophages was similar between the groups (J) (n=4-6, two technical replicates measured in duplicates, *p<0.05).

Abbreviation: TI = Test item;

Figure 4 shows evaluation of angiogenesis at the wound site by calculating the number of

CD31 stained blood vessels (A-D). The highest number was obtained in TI3 which was significantly different to control (E) (n=4-6, two technical replicates measured in duplicates). Abbreviation: TI = Test item;

Figure 5 illustrates neo-epithelium formation. The measured area is indicated as a red line (A-D). The length (E) was measured between the groups (n=4-6 and control n=11, 2 technical replicates measured in duplicates; *p < 0.05). Abbreviations: TI = Test item;

Figure 6 illustrates visual evaluation of layers of the proliferating cells in the epithelium from the PCNA-stained tissue sites, which are indicated with red squares (A-D). The layers and number of the stained cells were calculated (n=3-6, 2 technical replicates measured in duplicates) (E-F). Abbreviation: TI = Test item;

Figure 7 illustrates amount of collagen deposition in granulation tissue based on the

Masson Trichrome stained tissue sections (A-D). Color intensity showed a significant difference between TI3 to other treatment groups (n=4-6, control n=11, 2 technical replicates measured in duplicates) (E-F). The white arrow indicates the presence of NFC hydrogel (in A figure as an example). Abbreviation: TI =Test item.

DETAILED DESCRIPTION OF THE DISCLOSURE

External growth factors have gained a lot of attention in wound healing. As a topical treatment, single and multiple growth factor solutions have been studied. Platelet-rich plasma (PRP) is a source of growth factors that has been studied in wound tissue regenerative applications. PRP is generally referred to as high platelet concentrate (around & one million platelets/ul) from the autologous blood sample (around 200 000 platelets/ul),

N 25 obtained through centrifugation. Platelets release reguired bioactive compounds during

S wound healing, including platelet-derived growth factor (PDGF), epidermal growth factor

O (EGF), fibroblast growth factor (FGF), vascular endothelial growth factor (VEGF), and z interleukin-1 (IL-1). PRP has shown potential results in studies of various cutaneous > wound types, including acute wounds, burns, plastic surgery wounds, and chronic wounds.

O 30 Studies have reported PRP to enhance keratinocyte, fibroblast, and endothelial cell 2 functions, angiogenesis, and activation of macrophages during active inflammation.

I Furthermore, pre-clinical studies concerning allogenic options for PRP have been reported without any immunosuppressive effects. However, clinical results on efficacy of PRP are incongruous. One reason behind varying efficacy could be fast degradation of the bioactive compounds after administration due to proteases in the body. In addition, injected PRP can be dislocated and rapidly cleared from the wound site. To enhance the efficacy and stability of PRP and also single growth factor solutions, suitable carrier biomaterials are needed from which bioactive compounds could be released in a controlled manner.

As discussed above, the efficacy of bioactive compounds that promote wound healing is often diminished due to fast degradation and untargeted localization after transplantation.

For this reason, we have evaluated the potential of nanofibrillated cellulose (NFC) hydrogel as a carrier for PRP, that could be released to the wound site by degrading NFC with cellulase. In this study, we examined the effects of NFC hydrogel formulations including

PRP and cellulase on skin cells’ migration and proliferation via in vitro scratch wound model. The suitability for accelerated wound healing was studied in vivo in full thickness wound model in mice. None of the NFC hydrogel formulations disturbed normal cell behavior. Cellulase was successfully used to degrade NFC, and premature degradation could be prevented. Inhibition of NFC hydrogel towards fibroblast migration rate was observed. In vivo, NFC hydrogel showed enhanced neo-epithelization and supported collagen deposition. In addition, significantly induced angiogenesis was obtained via PRP release after degrading NFC hydrogel with cellulase without abnormal host reaction. This study proved the applicability of NFC hydrogel as a scaffold for PRP with a controlled release through cellulase degradation.

For the purposes of promoting an understanding of the principles of the invention, reference will now be made to certain embodiments and specific language will be used for describing the same. It will nevertheless be understood that no limitation of the scope of the invention is thereby intended, and alterations and modifications in the illustrated methods and agents, and further applications of the principles of the invention as illustrated 0 25 therein are herein contemplated as would normally occur to one skilled in the art to which < the invention relates. Unless defined otherwise, all technical and scientific terms used 2 herein have the same meaning as commonly understood by one of ordinary skill in the art

O to which this invention pertains. = As used herein, the term "wound" is used to refer broadly to injuries to the skin, mucous a 30 membrane, and subcutaneous tissue including tendons, initiated in different ways and with

O varying characteristics. Wounds are generally classified into one of four grades depending 2 on the depth of the wound: Grade I: wounds limited to the epithelium; Grade II: wounds

S extending into the dermis; Grade III: wounds extending into the subcutaneous tissue; and

Grade IV or full-thickness wounds: wounds in which bones are exposed e.g., a bony pressure point such as the greater trochanter or the sacrum.

As used herein, the term "chronic wound" refers to a wound that has not healed within 30 days. Examples of chronic wounds are neuropathic ulcers, pressure sores, venous stasis ulcers, and ulcers caused by diabetes and its' complications. As used herein the term "wound healing” or "cicatrisation” refers to an intricate process in which the skin or mucous 5 membrane or another organ-tissue repairs itself after injury. The classic model of wound healing is divided into three or four sequential, yet overlapping, phases: (1) hemostasis, (2) inflammatory, (3) proliferative and (4) remodeling phase. Within minutes post-injury, platelets (thrombocytes) aggregate at the injury site to form a fibrin clot, acting to control active bleeding (hemostasis). In the inflammatory phase, bacteria and debris are phagocytosed and removed, and factors are released that cause the migration and division of cells involved in the proliferative phase. In the proliferative phase angiogenesis, collagen deposition, granulation tissue formation, epithelialization, and wound contraction take place. In angiogenesis, new blood vessels are formed by vascular endothelial cells. In fibroplasia and granulation tissue formation, fibroblasts grow and form extracellular matrix by excreting collagen and fibronectin. Concurrently, re-epithelialization of the epidermis occurs, whereby epithelial cells proliferate and ‘crawl’ atop the wound bed, providing cover for the new tissue.

In an aspect, the invention relates to a nanofibrillar cellulose composition comprising nanofibrillar cellulose (NFC), cellulase and one or more bioactive agent(s).

As used herein, the bioactive agent(s) are substances that promote wound healing, and particularly are single growth factors and/or assortments of multiple growth factors such as PRP and/or bodies such as whole cells and extracellular vesicles that may excrete or produce single or multiple growth factors. The bioactive agent(s) may be selected from vascular endothelial growth factors (VEGFs) such as VEGF-A, VEGF-C and VEGF-D; 0 25 nerve growth factor (NGF); NGF-beta; platelet-derived growth factor (PDGF); fibroblast < growth factors (FGFs) such as FGF1, FGF2, FGF3, FGF4, FGF5, FGF6, FGF7, FGFS, 2 FGF9, FGF10, FGF11, FGF12, FGF13, FGF14, FGF15, FGF16, FGF17, FGF18, FGF19,

O FGF20, FGF21, FGF22 and FGF23; epidermal growth factor (EGF); tumor necrosis factor - (TNF); transforming growth factors (TGFs) such as TGF-alpha, TGF-beta 1, TGF-beta 2

E 30 and TGF-beta 3; insulin-like growth factors (IGFs) such as IGF-I, IGF-II and des(l-3)-lGF-

O I (brain IGF-I); neurotrophin-3 (NT-3); brain-derived neurotrophic factor (BDNF);

O interleukins (ILs) such as IL-1, IL-1 alpha, IL-1 beta, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8,

O IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, IL-18, IL-19, IL-20, IL-21, IL-22,

IL-23, IL-24, IL-25, IL-26, IL-27, 11-28, IL-29, IL-30, IL-31, IL-32, IL-33, IL-35 and IL-36; whole cells; extracellular vesicles; and platelet-rich plasma (PRP). The whole cells can be of any cell type, and they can include stem cells, undifferentiated cells, precursor cells, as well as fully differentiated cells and combinations thereof. In some embodiments, the whole cells comprise cell types selected from the group consisting of keratocytes, keratinocytes, fibroblast cells, epithelial cells and combinations thereof. In some embodiments, the cells are selected from the group consisting of stem cells, progenitor cells, precursor cells, connective tissue cells, epithelial cells, muscle cells, neuronal cells, endothelial cells, fibroblasts, keratinocytes, smooth muscle cells, stromal cells, mesenchymal cells, immune system cells, hematopoietic cells, dendritic cells, hair follicle cells and combinations thereof.

In some embodiments, the cells use the composition, particularly the hydrogel, as a scaffold to grow and/or migrate in the wound bed. A nutritional agent can be added to the composition to affect various activities or properties of the cells, such as cell growth and proliferation, cell adhesion, differentiation, migration, maintenance of undifferentiated states, secretion of extracellular matrix, and secretion of molecules, including growth factors, prostaglandins, cytokines and the like. The nutritional agents include vitamins, essential and non-essential amino acids, essential and non-essential fats and combinations thereof, hyaluronic acid, derivatives of hyaluronic acid, Aloe vera gel, propylene glycol, beta-1,3-glucan, and buffer substances for maintaining the pH of the composition in the range from 4 to 9.

One of the materials for forming the composition of the invention thus is nanofibrillar cellulose (NFC), also called nanocellulose, which refers to isolated cellulose fibrils or fibril bundles derived from cellulose raw material. Nanofibrillar cellulose is based on a natural polymer that is abundant in nature. Nanofibrillar cellulose has a capability of forming viscous hydrogel in water. Nanofibrillar cellulose production techniques may be based on 0 25 mechanically disintegrating fibrous raw material, such as grinding of aqueous dispersion < of pulp fibers to obtain nanofibrillated cellulose. After the grinding or homogenization 2 process, the obtained nanofibrillar cellulose material is a dilute viscoelastic hydrogel.

O The viscoelastic hydrogel may be an agueous gel at an NFC concentration of 0.1-3.5%

E (w/w), for example 0.2-3.5% (w/w). An example of commercially available nanofibrillar a 30 cellulose hydrogel is GrowDex® by UPM.

S In an embodiment, the nanofibrillar cellulose composition comprises NFC at a

N concentration of 0.1 to 3.5 % (w/w), preferably from 0.2 to 3.5 % (w/w). In one embodiment

N the NFC concentration is 0.2 to 1.0 % (w/w), preferably 0.5 to 1.0 % (w/w).

In a preferred embodiment, the NFC in the composition is in a form of a hydrogel.

The removal of cellulose nanofibers from the composition to release bioactive molecules such as growth factors contained therein may be carried out with enzymes using enzymatic degradation of cellulose molecules. Suitable enzymes are for example cellulases that are widely available commercially. Cellulases are able to hydrolyse [beta]-(1-4)-bonds in cellulose into monosaccharides such as B-glucose, or shorter polysaccharides and oligosaccharides. Based on the type of degradative reaction catalyzed, types of cellulases include endocellulases, exocellulases, cellobiases, exocellobiohydrolases, oxidative cellulases and cellulose phosphorylases. Total hydrolysis of NFC to monomeric sugars may also necessitate that the enzyme mixture contains endo acting hemicellulases, such as xylanases and mannanases, and beta-D-glycosidases, beta-D-xylosidases and beta-

D-mannosidases. The cellulase used in the nanofibrillar cellulose composition may comprise one type of cellulase or two or more types of cellulases, and/or be a mixture of several cellulases of the same type.

Upon hydrolysis of NFC hydrogel, the viscosity of the hydrogel is drastically lowered and the bioactive compound is released. The degradation product, glucose, is generally non- toxic to tissues.

The amount of cellulase present in the composition affects degradation rate of cellulose and therefore also release rate of the bioactive compound. We have determined that a concentration of 10 ug to 5000 ug cellulase per mg of cellulose, preferably 50 ug to 3000 ug cellulase per mg of cellulose is suitable for controlled release.

In an embodiment, the nanofibrillar cellulose composition comprises one or more cellulase(s) at a concentration of 10 ug to 5000 ug cellulase per mg of cellulose, preferably ug to 3000 pg cellulase per mg of cellulose. In one embodiment the nanofibrillar cellulose composition comprises one or more cellulase(s) at a concentration of 100 ug to

S 25 2000 pg cellulase per mg of cellulose, preferably 300 ug to 1000 ug cellulase per mg of

N cellulose. o <?@ Cellulose nanofibrils and/or cellulose nanocrystals may be prepared from cellulose raw

N material of plant origin. The raw material may be based on any plant material that contains

E cellulose. The raw material may also be derived from certain bacterial fermentation

AN 30 processes. In an embodiment, the plant material is wood. Wood may be from a softwood

S tree, such as spruce, pine, fir, larch, douglas-fir or hemlock, or from a hardwood tree, such

N as birch, aspen, poplar, alder, eucalyptus, oak, beech or acacia, or from a mixture of

N softwoods and hardwoods. In an embodiment, the cellulose nanofibril(s) is/are obtained from wood pulp. In an embodiment, the cellulose nanofibril(s) is/are obtained from hardwood pulp. In an example, the hardwood is birch. In an embodiment, the cellulose nanofibril(s) is/are obtained from softwood pulp.

Cellulose nanofibrils may be made of plant material. In an example, the cellulose nanofibrils are obtained from non-parenchymal plant material. In such a case, the cellulose nanofibrils may be obtained from secondary cell walls. One abundant source of such cellulose fibrils is wood fibres. The smallest cellulosic entities of cellulose pulp of plant origin, such as wood, include cellulose molecules, elementary fibrils, and microfibrils.

Microfibril units are bundles of elementary fibrils caused by physically conditioned coalescence as a mechanism of reducing the free energy of the surfaces.

Cellulose nanofibrils may be manufactured by homogenizing wood-derived fibrous raw material, which may be chemical pulp. Cellulose fibers may be mechanically disintegrated to produce fibrils which have a diameter in the nanometer range, which diameter may be up to 200 nm, or up to 50 nm, for example in the range of 1-200 nm or 1-100 nm, and gives a dispersion of fibrils in water. The cellulose nanofibrils may be type | cellulose. The fibrils may be reduced to a size in which the diameter of most of the fibrils is in the range of 2-20 nm. The fibrils originating from secondary cell walls may be essentially crystalline, with a degree of crystallinity of at least 55 %. Such fibrils may have different properties than fibrils originated from primary cell walls; for example, the dewatering of fibrils originating from secondary cell walls may be more challenging.

In the context of this specification, the term "cellulose nanofibrils” may refer to cellulose fibrils or fibril bundles separated from cellulose-based fiber raw material. These fibrils are characterized by a high aspect ratio (length/diameter): their length may exceed 1 um, whereas the diameter typically remains smaller than 200 nm. The smallest fibrils are in the scale of so-called elementary fibrils, their diameter being typically in the range of 2-12 nm.

S 25 The dimensions and size distribution of the fibrils may depend on the refining method and

N efficiency. Cellulose nanofibrils may be characterized as a cellulose-based material, in

S which the median length of particles (fibrils or fibril bundles) is not greater than 50 um, for

O example in the range of 1-50 um, and the particle diameter is smaller than 1 um, for = example in the range of 2-500 nm. In case of native cellulose nanofibrils, in an embodiment - 30 the average diameter of a fibril is in the range of 5-100 nm, for example in the range of 10-

O 50 nm. Intact, unfibrillated microfibril units may be present in the nanofibrils or the hydrogel 2 composition. In the context of this specification, the term "cellulose nanofibrils” is not meant

S to encompass non-fibrillar, rod-shaped cellulose nanocrystals or whiskers.

The term "cellulose nanocrystals" may be understood, in the context of this specification, to refer to non-fibrillar, rod-shaped cellulose nanocrystals. Cellulose nanocrystals are a highly crystalline material; it may be referred to as cellulose nanocrystals (CNC), nanocrystals of cellulose (NCC) or cellulose nanowhiskers (CNW). The nanocrystals are rod-like and stiff, have a narrow size distribution and are shorter than nanofibrils. The nanocrystals also have lower viscosity and yield strength and are typically not as good at holding water as nanofibrillar cellulose. Cellulose nanocrystals may have a width of about 2 - 30 nm. Cellulose nanocrystals may have a length of about 100 nm to several micrometers, or e.g. 100 - 250 nm. They may be obtainable or obtained by acid hydrolysis of cellulose fibers, whereby non-crystalline regions of the cellulose fibers may be selectively degraded. In the early stage of the hydrolysis, the acid may diffuse into the non- crystalline parts of the cellulose fibers and hydrolyze the glycosidic bonds. After these, more easily accessible glycosidic bonds in the cellulose fibers may be hydrolyzed. Finally, hydrolysis may occur at the reducing end groups and at the surface of the nanocrystals.

The nomenclature relating to cellulose nanofibrils is currently not uniform, and terms may be inconsistently used in the literature. For example, the following terms may have been used as synonyms for cellulose nanofibrils: cellulose nanofiber (CNF), nanofibril cellulose, nanofibrillar or nanofibrillated cellulose (NFC), nanocellulose, nano-scale fibrillated cellulose, microfibrillar cellulose, cellulose microfibrils, microfibrillated cellulose (MFC), and fibril cellulose.

Thus, a hydrogel comprising cellulose nanofibrils may refer to a nanofibrillar cellulose (NFC) hydrogel e.g. in this specification.

Cellulose nanofibrils are characterized by a large specific surface area and a strong ability to form hydrogen bonds. In water dispersion, the cellulose nanofibrils typically appear as either light or turbid gel-like material. Depending on the fiber raw material, cellulose nanofibrils may also contain small amounts of other wood components, such as & 25 hemicellulose or lignin. The amount is dependent on the plant source. a Different grades of cellulose nanofibrils may be categorized based on three main <?@ properties: (i) size distribution, length and diameter; (ii) chemical composition; and (iii)

N rheological properties. To fully describe a grade, the properties may be used in parallel.

E Examples of different grades may include native (or non-modified) cellulose nanofibrils,

AN 30 oxidized cellulose nanofibrils (high viscosity), oxidized cellulose nanofibrils (low viscosity), = and carboxymethylated cellulose nanofibrils. Within these main grades, also sub-grades & may exist, for example: extremely well fibrillated vs. moderately fibrillated, high degree of

N substitution vs. low, low viscosity vs. high viscosity, etc. The fibrillation technigue and the chemical pre-modification may have an influence on the fibril size distribution. Typically, non-ionic grades may have a wider fibril diameter (for example in the range of 10-100 nm,

or 10-50 nm), while the chemically modified grades may be thinner (for example in the range of 2-20 nm). The distributions of the fibril dimensions may be also narrower for the modified grades. Certain modifications, especially TEMPO oxidation, may yield shorter fibrils.

Depending on the raw material source, e.g. hardwood (HW) vs. softwood (SW) pulp, different polysaccharide compositions may be present in the final nanofibrillar product.

Commonly, the non-ionic grades are prepared from bleached birch pulp, which may yield a high xylene content (25 % by weight). Modified grades may be prepared either from HW or SW pulps. In such modified grades, the hemicelluloses may also be modified together with the cellulose domain. The modification may not be homogeneous, i.e. some parts may be modified to a greater extent than others. Thus, a detailed chemical analysis may not be possible - the modified products are typically complex mixtures of different polysaccharide structures.

In an aqueous environment, a dispersion of cellulose nanofibrils may form a viscoelastic hydrogel network. The gel may be formed at relatively low concentrations of, for example, 0.05-0.2% (w/w), dispersed and hydrated entangled fibrils. The viscoelasticity of the NFC hydrogel may be characterized, for example, by dynamic oscillatory rheological measurements. Cellulose nanofibril hydrogels may exhibit characteristic rheological properties. For example, they are shear-thinning or pseudoplastic materials, which means that their viscosity depends on the speed (or force) by which the material is deformed.

When measuring the viscosity in a rotational rheometer, the shear-thinning behavior is seen as a decrease in viscosity with increasing shear rate. The hydrogels show plastic behavior, which means that a certain shear stress (force) is required before the material starts to flow readily. This critical shear stress is often called the yield stress. The yield 0 25 stress can be determined from a steady state flow curve measured with a stress-controlled < rheometer. When the viscosity is plotted as function of applied shear stress, a dramatic 2 decrease in viscosity can be seen after exceeding the critical shear stress. The zero-shear

O viscosity and the yield stress may be the most important rheological parameters to - describe the suspending power of the materials. These two parameters may separate the

E 30 different grades guite clearly and thus may enable classification of the grades.

O Dimensions of the fibrils or fibril bundles may be dependent on the raw material and the 2 disintegration method. Mechanical disintegration of the cellulose raw material may be

S carried out with any suitable equipment such as a refiner, grinder, disperser, homogenizer, colloider, friction grinder, pin mill, rotor-rotor dispergator, ultrasound sonicator, fluidizer such as microfluidizer, macrofluidizer or fluidizer-type homogenizer. The disintegration treatment may be performed at conditions in which water is sufficiently present to prevent the formation of bonds between the fibers.

In an example, the disintegration is carried out by using a disperser having at least one rotor, blade or similar moving mechanical member, such as a rotor-rotor dispergator. One example of a rotor-rotor dispergator is an Atrex device.

Another example of a device suitable for disintegrating is a pin mill, such as a multi- peripheral pin mill. One example of such device is described in US 6202946 B1.

In an embodiment, disintegrating is carried out by using a homogenizer.

In the context of this specification, the term "fibrillation" may generally refer to disintegrating fiber material mechanically by work applied to the particles, whereby cellulose fibrils are detached from the fibers or fiber fragments. The work may be based on various effects, such as grinding, crushing or shearing, or a combination of these, or another corresponding action that reduces the particle size. The energy taken by the refining work may normally be expressed in terms of energy per processed raw material quantity, in units of e.g. kWh/kg, MWh/ton, or units proportional to these. The expressions "disintegration" or "disintegration treatment” may be used interchangeably with "fibrillation". The fiber material dispersion that is subjected to fibrillation may be a mixture of fiber material and water (or an agueous solution), also herein called "pulp". The fiber material dispersion may refer generally to whole fibers, parts (fragments) separated from them, fibril bundles, or fibrils mixed with water, and typically the agueous fiber material dispersion is a mixture of such elements, in which the ratios between the components are dependent on the degree of processing or on the treatment stage, for example number of runs or "passes" through the treatment of the same batch of fiber material. @ The disintegrated fibrous cellulosic raw material may be modified or nonmodified fibrous < 25 raw material. Modified fibrous raw material means raw material where the fibers are 2 affected by a modification treatment so that cellulose nanofibrils are more easily © detachable from the fibers. The modification may be performed to fibrous cellulosic raw

N material which exists as a suspension in a liquid, e.g. pulp.

E The modification treatment to the fibers may be chemical or physical. In chemical

O 30 modification, the chemical structure of cellulose molecule is changed by a chemical 2 reaction ("derivatization" of cellulose), for example so that the length of the cellulose

I molecule is not affected but functional groups are added to B-D-glucopyranose units of the polymer. The chemical modification of cellulose may take place at a certain conversion degree, which is dependent on the dosage of reactants and the reaction conditions, and often it is not complete so that the cellulose will stay in solid form as fibrils and does not dissolve in water. In physical modification anionic, cationic, or nonionic substances or any combination of these may be physically adsorbed on cellulose surface. The modification treatment may also be enzymatic. The cellulose in the fibers may be particularly ionically charged after the modification, because the ionic charge of the cellulose may weaken the internal bonds of the fibers and may later facilitate the disintegration to cellulose nanofibrils. The ionic charge may be achieved by chemical or physical modification of the cellulose. The fibers may have a higher anionic or cationic charge after the modification compared with the starting raw material. Commonly used chemical modification methods for making an anionic charge may include oxidation, where hydroxyl groups are oxidized to aldehydes and carboxyl groups, sulphonization and carboxymethylation. A cationic charge in turn may be created chemically by cationization by attaching a cationic group to the cellulose, such as a quaternary ammonium group.

The cellulose may be oxidized. In the oxidation of cellulose, primary hydroxyl groups of cellulose may be oxidized catalytically by a heterocyclic nitroxyl compound, for example 2,2,6,6-tetramethylpiperidinyl-1-oxy free radical, generally called "TEMPO". At least some of the primary hydroxyl groups (C6-hydroxyl groups) of the cellulosic B-D-glucopyranose units may be selectively oxidized to carboxylic groups. Some aldehyde groups may also be formed from the primary hydroxyl groups. The cellulose may be oxidized to a level having a carboxylic acid content in the oxidized cellulose in the range of 0.6-1.4 mmol

COOH! g pulp, or 0.8-1.2 mmol COOH / g pulp, for example to 1.0-1.2 mmol COOH/ g pulp, determined by conductometric titration. When the fibers of oxidized cellulose obtained in this manner are disintegrated in water, they may give a stable transparent dispersion of individualized cellulose fibrils, which may be, for example, of 3-5 nm in width. 0 25 Cellulose nanofibrils may also be characterized by the average diameter (or width), or by < the average diameter together with the viscosity, such as Brookfield viscosity or zero shear 2 viscosity. In an embodiment, said cellulose nanofibrils have a number average diameter © of a fibril in the range of 1-200 nm, preferably 1-100 nm, more preferably 2-100 nm. In an

N embodiment, the cellulose nanofibrils have a number average diameter of fibrils in the & 30 range of 1-50 nm. In an embodiment, the cellulose nanofibrils have a number average

S diameter of fibrils in the range of 2-15 nm, such as TEMPO oxidized nanofibrillar cellulose.

S The diameter of a fibril or fibrils may be determined using several techniques, such as by

O microscopy. Fibril thickness and width distribution may be measured by image analysis of the images from a field emission scanning electron microscope (FE-SEM), a transmission electron microscope (TEM), such as a cryogenic transmission electron microscope (cryo-

TEM), or an atomic force microscope (AFM). In general, AFM and TEM may be well suited for cellulose nanofibril grades with narrow fibril diameter distribution.

The viscosity of the cellulose nanofibrils or of the hydrogel composition may be measured using a rheometer. In an example, a rheometer viscosity of the nanofibrillar cellulose dispersion is measured at 22°C with a stress controlled rotational rheometer (AR-G2, TA

Instruments, UK) equipped with a narrow gap vane geometry (the vane having a diameter of 28 mm and a length of 42 mm) in a cylindrical sample cup having a diameter of 30 mm.

After loading the samples to the rheometer they are allowed to rest for 5 min before the measurement is started. The steady state viscosity is measured with a gradually increasing shear stress (proportional to applied torque) and the shear rate (proportional to angular velocity) is measured. The reported viscosity (=shear stress/shear rate) at a certain shear stress is recorded after reaching a constant shear rate or after a maximum time of 2 min.

The measurement is stopped when a shear rate of 1000 s-1 is exceeded. This method may be used for determining the zero-shear viscosity.

In one example, the cellulose nanofibrils, when dispersed in water, provide a zero shear viscosity ("plateau of constant viscosity at small shearing stresses) in the range of 1000- 100000 Pa-s, such as in the range of 5000- 50000 Pa-s, and a yield stress (shear stress where the shear thinning begins) in the range of 1-50 Pa, such as in the range of 3-15 Pa, determined by rotational rheometer at a consistency of 0.5% (w/w) by weight in agueous medium.

The cellulose nanofibrils may have a storage modulus in the range of 0.3 to 50 Pa, when dispersed to a concentration of 0.5 w% in water. For example, the storage modulus may be in the range of 1 to 40 Pa, or in the range of 1 to 20 Pa, or in the range of 2 to 10 Pa, when dispersed to a concentration of 0.5 w% in water. & 25 Turbidity is the cloudiness or haziness of a fluid caused by individual particles (total a suspended or dissolved solids) that are generally invisible to the naked eye. There are

ST several practical ways of measuring turbidity, the most direct being some measure of

N attenuation (that is, reduction in strength) of light as it passes through a sample column of

E water. The alternatively used Jackson Candle method (units: Jackson Turbidity Unit or

AN 30 JTU) is essentially the inverse measure of the length of a column of water needed to = completely obscure a candle flame viewed through it. & Turbidity may be measured quantitatively using optical turbidity measuring instruments.

N There are several commercial turbidometers available for measuring turbidity quantitatively. In the present case the method based on nephelometry is used. The units of turbidity from a calibrated nephelometer are called Nephelometric Turbidity Units (NTU).

The measuring apparatus (turbidometer) is calibrated and controlled with standard calibration samples, followed by measuring of the turbidity of the diluted NFC sample. In a turbidity measurement method, a nanofibrillar cellulose sample may be diluted in water, to a concentration below the gel point of said nanofibrillar cellulose, and turbidity of the diluted sample may be measured. The concentration in which the turbidity of the cellulose nanofibril samples is measured may be 0.1%. HACH P2100 Turbidometer with a 50 ml measuring vessel may be used for turbidity measurements. The dry matter of the cellulose nanofibril sample is determined and 0.5 g of the sample, calculated as dry matter, may be loaded in the measuring vessel, which may be filled with tap water to 500 g and vigorously mixed by shaking for about 30 s. Without delay the aqueous mixture may be divided into 5 measuring vessels, which are inserted in the turbidometer. Three measurements on each vessel may be carried out. The mean value and standard deviation may be calculated from the obtained results, and the final result may be given as NTU units.

One way to characterize cellulose nanofibrils or a hydrogel comprising them is to define both the viscosity and the turbidity. Low turbidity may correlate with a small size of the fibrils, such as small diameter, as small fibrils scatter light poorly. In general as the fibrillation degree increases, the viscosity increases and at the same time the turbidity decreases. This may happen, however, until a certain point. When the fibrillation is further continued, the fibrils may finally begin to break and cannot form a strong network any more.

Therefore, after this point, both the turbidity and the viscosity may begin to decrease.

In an example, the turbidity of anionic cellulose nanofibrils or of a hydrogel comprising them is lower than 90 NTU, for example from 3 to 90 NTU, such as from 5 to 60, for example 8-40, measured at a consistency of 0.1% (w/w) in aqueous medium, and 0 25 measured by nephelometry. In an example the turbidity of native cellulose nanofibrils or of < a hydrogel comprising them may be even over 200 NTU, for example from 10 to 220 NTU, 2 such as from 20 to 200, for example 50-200 measured at a consistency of 0.1% (w/w) in

O agueous medium and measured by nephelometry. To characterize the cellulose - nanofibrils or a hydrogel comprising them, these ranges may be combined with the

E- 30 viscosity ranges of the cellulose nanofibrils or of a hydrogel comprising them.

O In an embodiment, the nanofibrillar cellulose is medical grade nanofibrillar cellulose 2 comprising plant-based, such as wood-based, nanofibrillar cellulose. The medical grade

S nanofibrillar cellulose hydrogel ash a concentration of the nanofibrillar cellulose in the range of 0.1%-3.5% by weight, such as 0.2-3.5% by weight, the nanofibrillar cellulose having a number-average diameter of fibrils and/or fibril bundles of 100 nm or less, and a storage modulus in the range of 1-40 Pa determined by a rotational rheometer using plate geometry at a consistency of 0.5% by weight in agueous medium at 22+1*C. Alternatively the storage modulus may be presented with values measured by using vane geometry, which is in the range of 0.7-20 Pa. The medical grade nanofibrillar cellulose may be chemically and/or enzymatically unmodified.

Medical grade as used herein refers to material acceptable and suitable for use with living tissue, especially material which can be inserted, implanted and/or injected into a body or a tissue. Medical grade material is prepared at clean room conditions, wherein the quality of the materials throughout the manufacturing process is monitored and controlled, such as for microbial quality, purity and other related properties. For example, endotoxin content is controlled and maintained at low level. Medical grade nanofibrillar cellulose of the inveniton is safe, inert and biocompatible, which includes for example that it is non-toxic and it does not cause undesired reactions when in contact with living tissue, such as foreign substance reaction, allergic reaction, cytotoxic reaction, irritation or sensitization, or the like.

The medical grade nanofibrillar is substantially free of impurities and contaminants, or the impurities and contaminants may be under detection level. The medical grade nanofibrillar cellulose hydrogel preferably comprises bacterial endotoxins 5.5 EU/g (ml) or less determined according to Ph.Eur. 2.6.14.

In an embodiment, in the nanofibrillar cellulose composition NFC comprises native NFC, medical grade NFC and/or modified NFC, preferably medical grade NFC.

Auxiliary agents for enhancing the manufacturing process or improving or adjusting the properties of the product may be included in the nanofibrillar cellulose composition. Such n auxiliary agents may be soluble in a liguid phase of the composition, they may form an

S 25 emulsion or they may be solid. Auxiliary agents may be added already during the 6 manufacturing of the nanofibrillar cellulose composition to the raw material or they may be = added to a formed nanofibrillar cellulose composition. The auxiliary agents may be added

N for example by impregnating, spraying, dipping, soaking or the like. The auxiliary agents

E are usually not covalently bound to the nanofibrillar cellulose, so they may be releasable

AN 30 from the nanocellulose matrix. A controlled and/or sustained release of such agents may = be achieved along with the bioactive agent. Examples of auxiliary agents include & therapeutic (pharmaceutic) agents and other agents affecting the properties of the NFC,

N cellulase(s) or the bioactive agent(s), such as salts, buffers, surfactants, plasticizers, emulsifiers and the like. In one example the composition contains one or more salts, which may be added to enhance the properties of the final product or to facilitate physiological compatibility. Examples of salts include chloride salts, such as sodium chloride, calcium chloride and potassium chloride. The salt may be included in an amount in the range of 0.01-1.0% (w/w) by weight. The final composition may also be dipped or soaked in a solution of sodium chloride, such as in an aqueous solution of about 0.9% (w/w) sodium chloride. Desired salt content in the final composition may be in the range of 0.5-1% (w/w), such as about 0.9%, of the weight of the composition. The salts, buffers and the like agents may be provided to obtain physiological conditions.

Further auxiliary agents include proteins, peptides, carbohydrates, lipids, nucleic acids and fragments thereof, anti-viral compounds, anti-inflammatory compounds, antibiotic compounds such as antifungal and antibacterial compounds, cell differentiating agents, analgesics, contrast agents for medical diagnostic imaging, enzymes, cytokines, anaesthetics, antihistamines, agents that act on the immune system i.e. immunomodulators, hemostatic agents, hormones, angiogenic or anti-angiogenic agents, neurotransmitters, therapeutic oligonucleotides, viral particles, vectors, retinoids, cell adhesion factors, extracellular matrix glycoproteins, osteogenic factors, antibodies and antigens, steroids and painkillers.

The auxiliary agents can be in their free base or acid form, or in the form of salts, esters, or any other pharmacologically acceptable derivatives, enantiomerically pure forms, tautomers or as components of molecular complexes. The amount of bioactive agents in the composition can vary depending on the particular bioactive agent, the desired effect, and the time span for which the composition is to be administered.

The composition may optionally have a backing material attached thereto. In some embodiments, the backing material provides additional protection and/or support. In some embodiments, the backing is not permanent, and can be freely removable and can be

S 25 reattached, if needed. For example, in some embodiments, the backing can be removed

N by a health care provider to assess the progress of wound healing by inspecting the wound

S through the composition. In some embodiments, the backing may be in the form of a layer

O or more of cellulose (e.g., microbial or plant-based), a polyester, a polyurethane, a = polyethylene glycol or derivative thereof, a vinyl pyrrolidone acrylic, a methacrylic acid, a a 30 silicone isobutylene, a isoprene or a styrene or any combination thereof.

S The composition may optionally be incorporated in a support, such as gauze or non-woven

N material.

N In an aspect, the invention relates to the nanofibrillar cellulose composition comprising nanofibrillar cellulose, cellulase and one or more bioactive agent(s), wherein the composition is for use in wound treatment. As used herein, wound treatment refers particularly to dressing the wound with the composition of the invention. Generally, wound treatment involves steps such as cleaning, surgical examination and removal of dead skin, closing the wound and medical treatment of pain and/or infection. With the composition of the invention and its optional auxiliary agents, several of the wound treatment steps may be achieved such as dressing and closing the wound as well as treating pain and infection,

In another aspect, the invention relates to a wound treatment product comprising the nanofibrillar cellulose composition comprising nanofibrillar cellulose, cellulase and one or more bioactive agent(s). In a further aspect, the invention relates to use of the nanofibrillar cellulose composition comprising nanofibrillar cellulose, cellulase and one or more bioactive agent(s) for manufacturing a wound treatment product. The wound treatment product is particularly a wound dressing that may be a hydrogel composition and/or may have a backing material attached thereto or may be incorporated in a support.

In yet another aspect, the invention relates to a method of producing a nanofibrillar cellulose composition, comprising the steps of: a) providing a component comprising cellulase b) providing i) a component comprising NFC and one or more bioactive agent(s), or ii) a component comprising NFC and a component comprising one or more bioactive agent(s); c) combining i) the component comprising cellulase and the component comprising NFC > and one or more bioactive agent(s), or

O

N ii) the component comprising cellulase, the component comprising NFC and

S 25 the component comprising one or more bioactive agent(s). ©

N Thus, the method is a method of producing the composition of the invention described

E herein. Any embodiments related to the composition also apply to the method.

N In the method cellulase is thus provided as a component that is separate from NFC and/or

S bioactive agent(s). Providing cellulase separately ensures that its cellulose-degrading

O 30 effect ensues in a controlled manner upon preparing the composition which may then directly be used for example in wound treatment.

The method may further comprise a step of attaching a backing material to the composition or the component(s) of either step b)i) or b)ii), or a step of incorporating in a support the composition or the component(s) of either step b)i) or b)ii). Using the component(s) of either step b)i) or b)ii) in said further attaching or incorporating step facilitates combining cellulase to these components in step c) to initiate enzymatic cellulose degradation only at that point and not before.

In an embodiment, the component(s) of step b)i) or step b)ii) is(are) in a form of hydrogel.

In a further embodiment the component(s) of step b)i) or b)ii) is(are) in freeze-dried form.

Exemplary methods for freeze-drying NFC hydrogels are presented in EP4083113A1 and

EP4083112 A1, the contents of which are incorporated herein by reference.

Optionally, the method of the invention further comprises the step of b2) reconstituting the freeze-dried component to form a hydrogel. Step b2) is performed between steps b) and o).

The method of the invention may further comprise the step of d) mixing the combined components of step c) to produce an essentially homogeneous distribution of components.

In an aspect, the invention relates to a kit comprising: a) a container comprising cellulase; and b) i) a container comprising NFC and one or more bioactive agent(s), or ii) a container comprising NFC and a container comprising one or more bioactive agent(s).

In an embodiment, the kit is for use in the method described above. Also, it is disclosed use of the kit in said method.

O

N Employing separate containers for cellulase and NFC +/- bioactive agent(s) ensures that a the cellulose-degrading effect of cellulase does not ensue before the kit components are <Q 25 combined. This facilitates control of cellulose degradation and controlled release of ©

N bioactive agent(s).

I

= One or more of the kit component(s) may be incorporated or packed in a container that

N also acts as an application device, such as syringe, applicator, pump or tube containing

S the desired amount of the component(s). Said application device may comprise a

N 30 mouthpiece or nozzle providing constant flow of the component or composition in desired

N thickness and breadth and geometries. These "ready for use” devices can be packed, sterilized and stored, and used when desired.

Examples of wound and tissue types for which the composition of the invention may be used include, but are not limited to, skin wounds, burn wounds, chronic wounds, wounds in mucous membranes and ulcers as well as tendon wounds. Additional examples of wounds include wounds caused by laser surgery, radiation, chemical burns, cancer treatments, biopsy excision sites, pathogens, gunshot or knife stabbings, cosmetic surgery and reconstructive surgery and the like. Ulcers include neuropathic ulcers, pressure sores, venous stasis ulcers, and diabetic ulcers and the like. Suitably the composition may be used for the treatment of donor (sites that the physician uses for harvesting skin for grafting) and recipient sites in connection with therapy involving skin grafts, such as STSG and FTSG therapy. Said composition hydrogel may particularly suitably be used in grade

Il and grade III wounds, in partial thickness wounds (e.g., second degree burns, surgical wounds or wounds which still have the most of the dermis intact which can regenerate from the wound site) as well as in more severe wounds of grade IV. The grafts may be meshed or recruited minced skin grafting or epidermal sheet grafting may be used. The composition may also find use in connection with flap technique.

In skin grafts any known fixation techniques and agents may be used for fixing the graft and the composition may be applied directly to the graft without the need to use of dressings. However, if desired protective dressings may be used. In diabetic patients there are systemic challenges, such as the presence of neuropathy, endothelial dysfunction and increased susceptibility to infection, neuropathy because the patient is not in pain and often unaware of the severity of the wound until the infection spreads more proximally, often coupled with patient-specific obstacles, which make the care of chronic ulcerations in diabetic patients challenging. The composition may be used for improving the treatment of diabetic ulcers, particularly in connection with STSG therapy of chronic ulcers where n 25 sufficient hydration can be maintained, epithelialization is promoted, contraction is

S controlled and translocation of bacteria through it prevented. Other suitable applications of

O the composition are in dermal over grafting, where a STSG is applied to a recipient bed or = dermis or denuded scar tissue; in expanded grafts; in the treatment of unstable,

N depressed, corrugated or hypertrophic scars; in in the treatment of unstable or

E 30 hyperpigmented skin grafts, large pigmented nevi, radiation damage, vitiligo and removal

N of tattoos.

S

O Particularly complex skin injuries are caused by burns, which result in an extensive

O damage to the various skin layers. Burns are generally defined according to depth and range from 1* degree (superficial) to 3' degree (entire destruction of epidermis and dermis). The standard protocol of burn management highlights several factors which accelerate the process of optimal healing: (a) control of fluid loss; (b) barrier to wound infection; (c) fast and effective wound closure, optimally with skin grafts or skin substitutes; and (d) significant pain relief. The composition of the invention provides means for enhancing one or more of these factors. The compositions, suitably the ones comprising plant derived NFCs typically have remarkable high yield stress and high zero-shear viscosity at low concentrations. The compositions show shear-thinning behavior at higher shear rates, thus enabling easy dispensing of viscous compositions. When the compositions are sheared (e.g. in a rheometer or in a tube), the dispersed phase tends to move away from the solid boundaries, which leads to the creation of a lower-viscosity layer of liquid at the walls of the container. This phenomenon means that the resistance to flow, i.e. the viscosity is lower at the boundaries than in the bulk of the dispersion. Respectively, dispersing or injecting of the composition to the wound with an applicator, such as syringe or pipette is easy even at higher NFC concentrations (up to 3.5%), for providing an even and desired amount of the composition to the wound, even of more complicated configuration or shape. Thus, the composition can be applied even to irregular, small and otherwise complicated wounds and wound beds evenly. The phenomenon enables also easy dispensing of the composition with minimum disturbance of particles (bioactive agents etc.) dispersed in said composition.

The composition may have the potential to stop the bleeding of wounds (hemostasis), and can include agents that promote clotting, such as thrombin.

If desired the composition, applied to the wound site, may be covered by a secondary dressing, film or membrane.

The composition allows an excellent penetration to all contours of the wound and tissue and provides a proper moist environment. & 25 It also facilitates the process of necrotic debris removal (autolytic debridement), improves a the development of granulation tissue, accelerates the entire process of re-epithelialization

ST and angiogenesis, and helps in keeping the wound base clean. The composition may

N easily and gently be rinsed off with water from the injury or wound and replaced with a

E fresh composition if necessary.

S 30 The composition is inert, non-allergenic, anti-inflammatory, non-toxic, non-pyrogenic, and 2 promotes natural host cellular migration to a wound site.. Any of the embodiments

O discussed in this specification can be implemented with respect to a method, kit, or product.

The following examples are illustrative of embodiments of the present invention, as described above, and they are not meant to limit the invention in any way. The invention is illustrated also with reference to the figures.

EXAMPLES

Treatment formulations

Formulations included NFC hydrogel obtained from UPM Biomedicals, Finland (GrowDex®) with a fiber concentration of 1.5% (m/v), which was diluted to 0.8% with sterilized water in in vivo studies and 0.5% with the cell-specific medium in in vitro studies to obtain better imaging contrast. Qualified PRP plasma solution was obtained from

Finnish Red Cross Blood Service, which has been manufactured in clean rooms under

GMP and used as a cell culture supplement for ATMP production, primarily as described by Laitinen et al. (2016). PRP was thawed overnight in the fridge from -20 °C, after which it was spun down with a centrifuge (3500 rcf, 20 min) to collect PRP above the formed pellet. PRP was mixed with NFC hydrogel with a final concentration of 5% (v/v). Cellulase stock solution was obtained from UPM Biomedicals, Finland (Growdase™). The concentration was calculated according to product-specific instructions based on NFC hydrogel concentration (450 pg/ mg of Growdex®) and the stock solution was added directly to the formulation just before the experiments. In addition, 72 and V4 dilutions of the recommended cellulase concentration were studied in vitro with and without PRP to evaluate the NFC hydrogel degradation rate. Wound treatment formulations were NFC hydrogel (in vitro: NFC/ in vivo: Tl1), NFC hydrogel with PRP (in vitro: NFC+PRP/ in vivo:

TI2), and NFC hydrogel with PRP and cellulase solution (in vitro: NFC+PRP+C1,2,3/ in vivo: TI3). Pure 5% PRP diluted in cell specific medium was used as a positive control in in vitro studies.

O

N 25 Cell cultures

N

2 In vitro scratch wound model was provided to understand the effects of wound treatment © formulations on cells’ migration rate and proliferation. Three different cell lines were

N chosen based on their role in wound healing: Human skin keratinocytes (HaCaT, CAT:

T 300493, DKFZ cell line services, Heidelberg, Germany), primary human umbilical cord

S 30 endothelial cells (HUVEC, CAT: C-12203, PromoCell, Germany) and adult primary human 2 dermal fibroblasts (HDF, CAT: PCS-201-012, American Tissue Culture Collection, ATCC).

N HaCaTs were cultured in DMEM, high glucose, GlutaMAX (CAT: LT 61965026, Life

N Technologies, Carlsbad, CA, USA) supplemented with 10% of fetal bovine serum and used for experiments between passages 36-38. HUVECs were cultured in Endothelial cell medium 2 Supplement mix (CAT: BT C-2201, Biotop Oy, Finland) and used for experiments between passages 3-5. HDFs were cultured in fibroblast growth medium 2 (CAT:C-23020, PromoCell, Germany) and used for experiments between passages 3-7.

Culturing conditions were at +37 °C in a humified cell culture incubator with 5% CO.. The morphology and growth of the cells were evaluated with a Light microscope (Olympus

IX51).

In vitro scratch wound model

Cells were washed with x1 phosphate buffered saline without magnesium chloride (DPBS,

CAT: 14190144, Gibco, Waltham, MA, USA) and detached with TryPLE™ (CAT: 12563011, Gibco, Waltham, MA, USA) from the culture flasks and seeded into image-Lock 96-well plate (a*, CAT: 4379, Sartorius, Germany). Each cell type was cultured separately, with 30 000 cell density/well in 100 ul of cell medium. Cells were incubated in a cell incubator overnight, after which the cell medium was changed, and cell-free areas were created with Woundmaker (Incucyte®, CAT: 4563, Sartorius, Germany) according to the manufacturer's instructions. Briefly, a 96-well plate was placed on the base of the

Woundmaker and the lid with scratchers was placed on top of the cells by pressing it stably down. After the wound creation, cells were washed with cell medium three times after which 80 ul of cell medium was added, and cells were incubated in a cell incubator for 20 minutes. After the incubation, 20 ul of wound treatment formulation was added on top of the cell culture systems and imaged with Incucyte* S3 Live-Cell analyses instrument for 48 hours. The migration rate in the cell-free area was measured by calculating relative wound density using the Incucyte Scratch wound Analyses Software Module (CAT: 9600- 0012, Sartorius, Germany). In addition, the proliferation rate of the cells was studied by culturing the cells on a standard 96-well plate (CAT: 83.3925, Sarstedt, Numbrect, = 25 Germany) with 10 000 cell density/well. The protocol introduced above was used also for & proliferation experiments without wound making by adding wound treatment formulation 3 on top of the cultures. The proliferation was imaged and analyzed for 100 hours by

O calculating cell confluency using Incucyte Image Lock Module (Sartorius, Germany).

E Animals

N 30 18 female (9-10 weeks old) SKH1, specific pathogen-free mice (Crl:SKH1-Hrhr, SPF) were 2 used in the study. The animal experiments were approved by the Animal Care and Use

N Committee of the State Provincial Office of Southern Finland (license number

N ESAVI/12977/2022) and all the studies conformed to the following guidelines: Act on the

Protection of Animals Used for Scientific or Educational Purposes (497/2013),

Government Decree on the Protection of Animals Used for Scientific or Educational

Purposes (564/2013), Guidance document on the Recognition, Assessment and Use of

Clinical Signs as Humane endpoints for Experimental Animals Used in Safety Evaluation,

Environmental Health and Safety Monograph Series on Testing and Assessment (No 19.

OECD 2000).

The experiments were implemented in a GLP-certified central animal laboratory (BioCity,

Turku University, Finland) and the acclimatization period before the experiments was 13 days. The mice were housed 4-5 animals in one cage before the experiments and individually during the experiments starting on study day 0. Cellulose paper and cardboard houses were used as environmental enrichment in the cages. Laboratory room temperature was at +21 °C +/- 3 °C, with relative humidity ranging from 40-60% and lightning was artificial with 12 hours of light and 12 hours of dark. A standard laboratory diet (Teklad2920) and water were offered on an ad libitum and the animals were taken care of according to the standard operating procedures of Animal Laboratory. All the animals were randomized to study groups depending on the used treatment formulation (TI (test item) 1, 2, or 3) at study day 0. The clinical status was checked twice a day during the whole experiment period between 10-13 hours intervals. The animals were weighed on study day 0 and at the end of the study on day 9. In addition, mice were weighed regularly during the experiment period on days 1-8 to control animal welfare but the data is not compared with the days 1 and 9 due to the extra weight of the applied dressing.

When required, the outer dressing was replaced with a new one leaving the inner dressing layer untouched. On study day 0-2 mice were treated with a non-steroidal anti- inflammatory drug (16 mg/kg Rimadyl vet, Zoetis) twice a day with 10-13 hours intervals as a postoperative treatment. 0 25 Surgical procedure

N Mice were anesthetized (isoflurane 3.5%, Attane Vet 1000 mg/g) for creation of full- 3 thickness wounds under aseptic conditions. To prevent possible pain during the operation,

O mice were dosed with local anesthesia + NSAID + opioid combination (Lidocain 4 mg/kg = (Baxter), Bupag multidose vet 0.1 mg/kg (Ritchter Pharma) and Rimadyl vet 16 mg/kg * 30 (Zoetis)). As a postoperative treatment, Rimadyl was administered 6-8 hours after surgery

O and every 10-13 hours on study days 1 and 2. The surgical sites were disinfected with a 2 skin disinfectant and incisions were made on both sides of the mouse s back with sterile

S scissors and tweezers (Figure 1A). One wound was treated with one of the treatment formulations (Tl1, TI2, or TI3) and another wound was treated with a physiological sodium chloride solution (0.9% NaCl) as a control (Figure 1B). Approximately 50 ul of the formulation was placed on the wound site by pipet immediately after wound creation and wound measurement. A total of 6 mice were treated per one treatment formulation. After dosing, wounds were covered with transparent, non-occlusive polyurethane film coated with polyacrylate adhesive (Raucodrape®, Lohmann & Rauscher).

Wound monitoring and sampling

The length and width of the wounds were measured right after their creation and on study day 9, before the necropsy, by a calibrated digital caliper (Mitutoyo 0-150 mm). The wound area was calculated with equation [1] according to Moreira et al. (2015), and the percentage of the wound contraction was calculated according to equation [2]: (Length/2) x (width/2) x n [1] ((Area of the wound at study day 9)/area of the wound at study day 0)) x 100% [2]

The end of the study was determined to be wound closure on study day 9, during which mice were weighed and all macroscopic abnormalities at the wound site were recorded.

Blood samples (ca. 600 ul) were taken into K2E Microtainer& tubes for hematological analyses by heart puncture under isoflurane anesthesia. Hematology tests are listed in supporting information, Table S3, according to the protocol of UCTUCAL on VetScan HM5 hematological analyses. A piece of skin (1 cm x 1 cm) including the wound site was removed and placed into 4% buffered formalin. Subseguently, tissue samples were embedded in paraffin and cut into 4 um sections for further analyses.

Histopathology and immunohistochemistry

Cut sections were stained with Hematoxylin and Eosin stain (HE) to analyze the abundance of inflammation infiltrate, immune cells, or giant cells and measure the e formation of neo-epithelium. Masson's Trichrome staining was implemented to evaluate

S collagen deposition based on the blue color intensity. 2 25 Immunostaining was implemented according to the protocol by Koivuniemi et al., (2021). © Briefly, after deparaffinization and antigen retrieval, tissue sections were blocked of

I endogenous peroxidase activity in 3% H2O2 and nonspecific staining blocking was a implemented for 1 hour at room temperature (RT) in a humid chamber with 5% bovine

I serum albumin (BSA, Merck, Darmstadt, Germany) in tris buffered saline with tween 20

O

O 30 (TBS-T). Then after samples were blocked with endogenous biotin (Avidin/Biotin blocking

O kit, Vector Laboratories, Burlingame, CA, USA) for 15 + 15 minutes. Tissue sections were incubated with anti-rabbit CD31/ platelet endothelial cell adhesion molecule (PECAM-1, 1:500, CAT: NB100-2284, Novus Biologicals, Abingdon, UK,), anti-rabbit, proliferating cell nuclear antigen (PCNA, F-2, 1:500, CAT: SC-25280, Santa Cruz Biotechnology Inc.,

Dallas, TX, USA), ionized calcium-binding adaptor molecule 1 (lba-1, CAT: 019-19741,

FujiFilm, USA) or lysozyme (CAT: A0099, Agilent, Santa Clara, CA, USA) as a primary antibody in 3% BSA/PBS-T overnight at +4 °C in a humid chamber. After washing with

TBS-T, tissue sections were stained with biotinylated secondary antibody, goat anti-mouse

IgG (1:1000, Abcam, Cambridge, UK) in 3% BSA/TBS-T for 1 hour in a humid chamber.

VECTASTAIN Elite ABC reagents (Vector Elite) were used for antibody detection by staining the tissue sections for 30 minutes at RT after which they were treated with 3,3'- diaminobenzidine (DAB) HRP substrate treatment (Vector Elite) until desired staining density. Sections were counterstained with hematoxylin, dehydrated, and subseguently covered with cover clips using a mounting medium. To stain NFC hydrogel from the wound sites, tissue sections were stained with Alcian blue (1% in 3% acetic acid pH 2.5, CAT:

B8438-250ML; Sigma) for 15 minutes after deparaffinization and washing with ultrapure water. Then after sections were washed and stained with Counterstain, Nuclear Fast Red (CAT: 1.00121.0500, Merck) for 5 minutes. Subsequently, sections were washed, dehydrated, and mounted with Coverquick 2000.

All the tissue sections were scanned with Pannoramic 250 Flash III brightfield digital slide scanner. Thickness and length measurements were implemented with CaseViewer (3DHistech Ltd., version 2.4.). The number of newly formed blood vessels and proliferating cell layers were manually calculated, and the intensity of the stains was evaluated utilizing object and pixel classifications in QuPath 0.4.x software (version 0.4.3.).

Immunofluorescence

Sections for the immunofluorescence were deparaffinized and blocked with 5% BSA in

PBS/Tween 20 followed by incubation at +4 *C overnight with anti-rabbit CD31/ PECAM-

S 25 1 antibody (1:500) and Ki67 antibody (1:800, Abcam, Cambridge, UK) in 3% BSA/PBS-T.

N Subsequently, secondary antibodies, donkey anti-rabbit IgG Alexa Fluor 594 (1:500, Life 3 Technologies, USA) and goat anti-mouse Alexa 488 (1:500, Invitrogen, Carlsbad, CA,

O USA) were applied in 5% BSA/PBS-T with an incubation time of 3 hours at room

E temperature. Prolong Diamond Antifade Mountant with DAPI (Thermo Fischer Scientific) a 30 was used as the mounting medium and the slides were imaged with Leica DM6000B.

O Double-stained cells were evaluated with a QuPath object classification analyzer. 3 &

Statistical analyses

The data is presented as mean standard deviation (SD). Statistical significance was measured with one-way ANOVA and Tukey HSD post hoc test. Significance was concluded when p<0.05.

Results

Correlation between the degradation of NFC hydrogel and effects of formulations on cell migration in vitro

The effects of wound treatment formulations on migration and proliferation of HaCaTs,

HUVECs, and HDFs were evaluated utilizing Incucyte® Live Cell analyses (Table 1).

Relative wound density measurements showed cells treated with pure PRP reaching over 90% confluency the fastest after 24 hours. However, HaCaTs showed a slower migration rate in the presence of PRP till 12 hours when compared to other cell lines. NFC hydrogel alone slightly enhanced the migration rate in the case of HUVECs and HaCaTs, when compared to the control, and did not show significant changes for PRP properties.

Surprisingly, in the case of HDFs NFC hydrogel slowed down the migration rate when compared to the control and confluency was significantly different when compared to pure

PRP at timepoint 24 hours. NFC+PRP-treated HDFs showed similarities in the migration rate with the control. No significant differences were obtained between the groups and control in any cell line after 36 hours showing relative wound density to be >80% in every group, so the only differences observed were in migration rate during the experiment.

When cellulase was present, differences in the migration rates in cells treated with

NFC+PRP were observed (Table 2). Compared to the control, significantly higher migration rates for 24 hours were observed in NFC+PRP-treated HaCaTs and HDFs when @ higher concentrations of cellulase (C1, C2) were used. Similar effects of cellulase were < 25 observed also in the case of HUVECs, but the differences between the migration rates 2 were insignificant. The proliferation of all the cells was enhanced when NFC hydrogel with © and without PRP was added, and 100% confluency was reached faster compared with the

N control in every cell line. [an a

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Table 1. Relative wound density of HUVECs (*NFC+PRP vs control), HaCaTs (*Control vs NFC; NFC vs PRP), and HDFs (*12 h timepoint: NFC vs NFC+PRP; NFC vs PRP;

Control vs PRP; *24 h timepoint: NFC vs PRP). Cells were treated with NFC, NFC + PRP, or PRP (done in quadruplicates, *p< 0.05). Abbreviations: HUVEC = Human umbilical cord vein endothelial cell, HaCaT = Human keratinocytes, HDF = Human dermal fibroblasts,

NFC = Nanofibrillated cellulose, PRP = Platelet-rich plasma, RWD = Relative wound density, SD = standard deviation.

NFC+PRP | PRP

TIME | RWD RWD RWD RWD

SD SD SD SD he | [Te |e |e 4] 18] 22] 300] 0] 382] 218] 113] 44] [PB] 302] 68] 561] 148] 523] 164] 302] 23 = 96] 921] 68] ®0| 159] 989] 56] se] 25] 101.0

HDF | Contol | NFG_ | NFC+PRP | PRP

TIME | RWD RWD RWD RWD

SD SD SD SD

"mes elmetmeme 4] 22] 52] 60] 51] 218] 115] 44] 12]

I PR] 68] 74] 148) 43] 164] 86] 23] 26 36] 68] 103] 159] 35] 56] 70] 23] 45)

HUVEC NFC+PRP | PRP

TIME | RWD RWD RWD RWD

SD SD SD SD

"meles |e |e n of of of of of of of of 0]

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Table 2. Relative wound density of HUVECs, HaCaTs (*12 h timepoint: Control vs

NFC+PRP+C2; *24 h timepoint: Control vs NFC+PRP+C1/C2) and HDFs (*4 h timepoint:

NFC+PRP+C1 vs NFC+PRP+C3; *12 h timepoint: NFC+PRP+C1 vs NFC+PRP+C3;

NFC+PRP+C2 vs NFC+PRP+C3). Cells were treated with NFC+PRP with and without cellulase in different concentrations (C1 refers to the highest cellulase concentration 36% (v/v), C3 refers to the lowest cellulase concentration). (Done in guadruplicates, *p< 0.05).

Abbreviations: HUVEC = Human umbilical cord vein endothelial cell, HaCaT = Human keratinocytes, HDF = Human dermal fibroblasts, NFC = Nanofibrillated cellulose, PRP =

Platelet-rich plasma, C = Cellulase, RWD = Relative wound density, SD = standard deviation.

NFC+PRP+C1 | NFC+PRP+C2 | NFC+PRP+C3

TIME | RWD RWD RWD RWD

SD SD SD SD

BENEN

I Bj 302] 68] 497] 158) 618] 47] 432] 91] = 24] 777] 173] 998] 09] 900] 52] 364] 48] = 36] set] 68] 987] 08] 1007] 06] 1052 1018 1003

HDF | — Control — | NFC+PRP+C1 | NFC+PRP+C2 | NFC+PRP+C3

TIME | RWD RWD RWD RWD

SD SD SD SD

"6 ee | [Te | 2 | e

I e] 68] 74] 691] 158) 569] 59] 344] 127] 36] 68 103] 965] 26] 918] 36] 618] 109]

Q HUVEC NFC+PRP+C1 | NFC+PRP+C2 | NFC+PRP+C3

O

N TIME | RWD RWD RWD RWD

SD SD SD SD

3 mmm | ga | [Te m” - | 0] of of of of of of of o

N z - e 2 | 48] 976| 24] 985] 55] 996| 34] 974| 54

N

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Treatment formulations self-detached from the wound site and did not show abnormal wound contraction in vivo

All animals survived in good condition and no abnormal clinical signs, prolonged wound inflammation, or pain were observed during the experiment period. Weight development was negative in all mice. The weight loss was between 0.21-4.68 g, without significant differences between the treatment groups. Weight loss was however not considered to affect mice's well-being.

All wounds were closed on study day 9. Visual observations revealed formulation self- detachment from some of the closed wounds when NFC hydrogel was present (TI1, TI2), which was seen as a dried piece next to the wound or non-attached on top of the wound (Figure 2A-B). In the case of TI3, no remaining formulation was observed (Figure 2C).

Wound contraction was evaluated by measuring the wound size at the beginning and the end of the study. Based on the percentual wound contraction analyses, all the formulations showed slightly higher but insignificantly different contraction rates compared to the control. Wound contraction was 29.8-32.5% (+/- 4-7.8%) in the formulation-treated wounds and 39.8% (+/- 12.4%) in the control (Figure 2D).

All wounds showed normal cell infiltration without abnormal inflammation

On day 9 at the end of in vivo experiments, whole blood samples were taken from all the mice before they were sacrificed. Due to technical difficulties, some blood was collected from the thoracic cavity after aortic dissection, for which reason these samples were coagulated and were not analyzed further. Based on the lymphocyte and monocyte detections from the hematological analyses, none of the mice showed any acute inflammation. In addition, the mean platelet value difference was insignificant in all mice n when compared to reference values found in the literature. Hematological analyses did not

S 25 show differences between the treatment groups. 2 The histopathological evaluation between the treatment groups observed in HE and © Masson Trichrome staining was evaluated by the pathologist. All the treatment groups and - the control showed mild to moderate hyperplasia at the epidermis referring to the typical

T healing process. No differences in the maturation state of the connective tissue were

S 30 obsetved in which fibroblasts were oriented longitudinally. Cell infiltration consisting mainly 2 of macrophages was mild to moderate in the case of TI1 and control. In the case of TI2

O and TI3, macrophage aggregation was observed to be stronger around NFC hydrogel or possible NFC hydrogel remnants. NFC hydrogel was observed in some of the macrophages in TI1 and TI2 groups. Only some/singular multinucleated cell formation was observed in the treatment groups relating to mild natural local reaction to external material.

In addition, no encapsulation or fibrosis was observed around NFC hydrogel.

Immune cell infiltration was evaluated in more detail for macrophages. Based on the Iba- 1 staining (left-sided images, Figure 3A, C, E, G), which stains macrophages apart from their activity, TI3 showed significantly higher stain intensity (19%) when compared to other groups (<15%) (Figure 31). However, when tissue sections were stained with lysozyme, revealing active macrophages, no differences between the treatment groups were observed (right-sided images, Figure 3B, D, F, H) and the stained area of the total wound was under 1% in all treatment formulations and control (Figure 3J).

Release of PRP enhanced angiogenesis

Angiogenesis was evaluated by calculating the number of blood vessels stained with

CD31. TI3 showed a significantly higher number of blood vessels when compared to the control (Figure 4). A slight increase was also observed in TI1 and TI2 treatment groups.

However, in TI2 the number of blood vessels between tissue samples varied more than in other groups. The orientation of the vessels was visually evaluated whether they were towards epithelium or parallel to the wound, and both orientations were presented in every treatment group without significant differences.

To evaluate further whether endothelial cells forming the blood vessels were actively proliferating, fluorescence double staining with CD31 and Ki67 was implemented. Based on the Qupath pixel analyses, the number of double-stained endothelial cells was insignificant between the groups.

Enhanced formation of neo-epithelium and supported collagen deposition in the presence of NFC hydrogel

O To evaluate the guality of wound healing further, HE-stained tissue sections were used to

N 25 measure newly formed epithelium at the wound site (Figure 5A-D). All the treatment groups

S showed longer neo-epithelium than the control and a significant difference was observed

O in TI1 (Figure 5E).

E PCNA staining was used to evaluate actively proliferating keratinocytes in the epithelium

N (Figure 6A-D), and slight differences in the proliferating cell layers could be observed = 30 (Figure 6E). In the case of TI3, proliferating cells were located in three different cell layers

SG when compared to other treatment groups and control, which showed cells to be located

N in one to two layers. The total number of proliferating cells in the epidermis did not differ between the groups (Figure 6F).

Granulation tissue formation was analyzed based on Masson Trichrome staining, from which the blue color intensity of the granulation tissue was evaluated (Figure 7A-D). The blue color intensity resembling the amount of granulation deposition at the wound site showed differences (Figure 7E). The highest intensities were present in Tl1 and TI2 while

TI3 showed the lightest color meaning the lower presence of total collagen deposition. The presence of NFC hydrogel, that could be observed also from the Masson trichrome staining as light blue areas, was stained to evaluate whether any leftovers of the hydrogel were present in TI3, and no stained areas were observed when cellulase was present.

Discussion

We studied the potential of animal-free and non-toxic NFC hydrogel to be utilized as a suitable carrier for tissue regenerative PRP in acute deep wound healing. The release of

PRP and the final degradation of the hydrogel could be controlled with cellulase. Results revealed enhanced effects of NFC hydrogel on re-epithelization and supported natural collagen deposition. In addition, angiogenesis was significantly induced after the release of PRP. To our knowledge, in this work, the functionality of cellulase to control the compound release from the matrix and degradation of NFC hydrogel in vivo was shown for the first time.

Among the immune cells, keratinocytes and fibroblasts are one of the main cell types during wound healing, by taking part in neo-epithelization and extracellular matrix formation. In addition, sufficient nutrient and oxygen exchange is enabled by endothelial cells, which take the leading role in blood vessel formation. For this reason, the evaluation of the effects of wound treatment formulations on cell migration and proliferation functionalities is reguired. As expected, PRP had enhancing effects on cell migration and proliferation probably due to the presence of growth factors, especially PDGF (participates

S 25 in vessel maturation and activation of fibroblasts), VEGF (induction of angiogenesis), FGF

N (activates fibroblasts), and HGF (activates epithelial cells, and keratinocytes). Even

S without the presence of PRP, NFC hydrogel enhanced the proliferation of all the cells and

O migration of HUVECs and HaCaTs. This could be due to the highly organized, and = chemically inert 3D structure of the hydrogel which has proved its suitability already in - 30 former cell culture applications. Differing from other studied cell lines, migration of the

O HDFs was slower with NFC hydrogel when compared to the control. The same 2 phenomenon was observed in a study by Nuutila et al. 2018 in which NFC hydrogel was

S studied to significantly inhibit the migration of fibroblasts when evaluated via collagen sheet assay. Nuutila et al., 2018 also studied the effects of NFC hydrogel on in vivo wound contraction in pigs revealing significant inhibition. It is hypothesized that one reason for inhibiting the effect could be glucose units forming the NFC fibers since high glucose is shown to have unfavorable effects on wound closure. However, it must be noted that in

NFC hydrogel, glucose is located in the organized polymer and not as free molecules which differs from the situation in the hyperglycemic conditions. Furthermore, unlike in

Nuutila's research, NFC hydrogel did not show inhibiting effects on in vivo wound contraction in our study. The reason behind this could be the lower concentration of the used NFC hydrogel (0.8%) relating to lower mechanical stiffness since the inhibiting effects towards wound contraction were shown for NFC hydrogel with 1.7% fiber concentration.

The dilution could also explain the absence of the encapsulation of the material, which was shown in some samples in the previous study Koivuniemi et al. 2021, in which 1.5% native

NFC hydrogel was used for full-thickness wound treatment in mice. However, no pathological immune response towards the 1.5% NFC hydrogel was observed. In this study, 0.8% NFC hydrogel was successfully transplanted and observed at the wound site after 9 days of the experiment indicating the potential for localized administration of the hydrogel even at lower fiber concentration. In this sense, NFC hydrogel is a highly beneficial material since the mechanical stiffness can be easily adjusted for the desired applications. Another reason for which NFC hydrogel did not show inhibition of the contraction in our study could be that instead of pigs we used a mouse model, which has highly different wound closure mechanisms compared with humans.

Along with PRP, which is known for its re-epithelizing and collagen deposition effects, NFC is reported to be the suitable matrix for epithelization and even preserves the elasticity of the skin when studied in a dressing form. In our study, NFC hydrogel showed significantly stronger effects on neo-epithelization when compared to the control. Furthermore, the amount of collagen deposition was supported when NFC hydrogel was present with (TI2) n 25 and without PRP (TI1). Surprisingly, the amount of collagen deposition was the lowest in

S the case of TI3 including cellulase. One hypothesis could be again in the increased amount

O of free glucose molecules at the wound site after NFC degradation, which is known to be = an inducing factor in collagen metabolism due to the induced activity of proteolytic

N enzymes. All in all, the difference between the collagen deposition in control and TI3 was

E 30 insignificant, which concludes that none of the treatment groups affected harmfully on

N collagen formation.

S

O The potential of the utilization of cellulase was highlighted in blood vessel measurements,

O in which TI3 showed to enhance angiogenesis significantly. Angiogenesis refers to the new capillary formation from the already existing blood vessels enabling sufficient oxygen and nutrient exchange at the wound site. Angiogenesis can be induced with angiogenic factors, including VEGF and PDGF, for which reason results could be related to the release of PRP from the NFC hydrogel via cellulase actions. The functioning of cellulase could be also observed from the epithelial cell proliferation analyses in which TI3 showed to activate proliferating cell layers. Along with TI3, angiogenesis was slightly enhanced with TI1 and -2, in which vascularization was observed inside NFC hydrogel, relating to the biocompatibility of these formulations. However, in the case of TI2, increased variation between the parallel samples in blood vessel measurements was observed. This could relate on the differing release profiles of different bioactive compounds through diffusion from the NFC hydrogel. In general, growth factor release from hydrogels is mainly based on diffusion, which can be altered by the porous size and distribution. In addition, although native NFC hydrogel is known as chemically bioinert, it has been reported to have a slight negative charge, which can increase the release rate of negatively charged growth factors through repulsive forces. Better controllability in drug release applications has been obtained by degrading the carrier material, which highlights even more the potential of the use of cellulase in our case. In summary, the results suggest the suitability of NFC hydrogel for future angiogenic applications and correlated with our previous study in which adipose- derived stromal cells in vitro showed enhanced stimulative effects towards vascularization when they were 3D cells cultured in 0.125% NFC hydrogel. Furthermore, NFC in a dressing form has previously shown enhancing vascularization effects in skin graft donor site treatment, which supports our findings of the material in this study.

One factor affecting enhanced angiogenesis could be the presence of macrophages.

Infiltration of the immune cells is required during wound healing to phagocytose injured and dead cells at the tissue site and eliminate possible pathogens. In addition, these cells, including neutrophils, leucocytes, and macrophages, take part in chemotactic signaling, n 25 which is required in the progression of tissue regeneration. Macrophages are often related

S only to active inflammation state and immune defense, but research has revealed

O macrophage phenotypes and their role also in tissue regeneration. Macrophages secrete = growth factors, cytokines, and chemokines, and this paracrine signaling can take part in

N the activation of angiogenesis. TI3 revealed a significantly higher presence of

E 30 macrophages in Iba-1 staining, which is a protein expressed in macrophages. This could al correlate with the results of enhanced blood vessel formation. The reason for the higher

S infiltration of macrophages could be the released PRP, which is known to recruit

N macrophages to the wound site. For instance, in a previous study PRP showed increased

N infiltration of macrophages to the full-thickness wound in a tendon in mice, and depending on the leucocyte concentration in PRP angiogenesis was enhanced. This could be probably due to the higher presence of M2 phenotype, which is known as tissue regenerative macrophage. The other reason could be the foreign material reaction towards cellulase, which however did not show abnormal inflammation activity. Furthermore, macrophages tended to accumulate more strongly in specific areas and NFC hydrogel was observed to be degraded into multiple pieces. One possible explanation for this could be the ability of macrophages to degrade NFC hydrogel and/or cellulase. Studies have suggested that biomaterials, such as scaffolds could be degraded by macrophages through released enzymes and reactive oxygen species, and for larger particles by giant cell formation. It is believed that biomaterial microarchitecture could have a role in macrophage activity and even phenotype, for instance, the pore size of the material. In our study, mild observations of the giant cells were made, which related to normal foreign body reactions, but the degradation and phagocytosis ability of macrophages towards the treatment formulations needs to be studied more. To reveal that no abnormal or inflamed macrophage activity was present, lysozyme was used, which is a marker expressed in macrophages with biosynthetic activity. Lysozyme staining showed insignificant differences between the treatment groups and the control.

One of the main challenges in proper wound care is excess scar formation and attachment of the topical treatment to the wound site causing pain and even secondary wound formation. PRP and release of growth factors, like FGF, and EGF are discussed to be potential agents in scar management by enhancing collagen organization, for which reason stable release of PRP to the wound site could be advantageous. In addition, in our study treatment formulations including NFC hydrogel showed self-detachment from the wound site on study day 9. This supports the data obtained from the studies regarding

NFC-based dressing for skin-graft donor site treatment, in which the dressing was self- detached after wound healing increasing the compliance with the treatment. TI3 showed n 25 complete clearance of the NFC hydrogel, which could replace the need for subseguent

N formulation removal from the wound site. Furthermore, with the presence of cellulase, the a biodegradability of NFC hydrogel could be enabled even with controllable degradation = time, which was studied in vitro. The results showed promising data in increasing the

N degradation rate of NFC hydrogel with increasing cellulase concentration without harming

E 30 cell behavior. This property could be utilized in the future to control the PRP release and

N NFC hydrogel clearance. This study showed the potential of NFC hydrogel to be utilized 2 as a biocompatible carrier for PRP to be administered to the cutaneous wound site, which

N could be stably released utilizing cellulase following accelerated wound healing.

N

REFERENCES

Laitinen A, Oja S, Kilpinen L, Kaartinen T, Möller J, Laitinen S, et al. A robust and reproducible animal serum-free culture method for clinical-grade bone marrow-derived mesenchymal stromal cells. Cytotechnology 2016;68. https://doi.org/10.1007/s10616-014- 9841-x.

Moreira C, Cassini-Vieira P, da Silva M, Barcelos L da. Skin Wound Healing Model -

Excisional Wounding and Assessment of Lesion Area. Bio Protoc 201555. https://doi.org/10.21769/bioprotoc.1661.

Koivuniemi R, Xu O, Snirvi J, Lara-Säez I, Merivaara A, Luukko K, et al. Comparison of the Therapeutic Effects of Native and Anionic Nanofibrillar Cellulose Hydrogels for Full-

Thickness Skin Wound Healing. Micro 2021;1. https://doi.org/10.3390/micro1020015.

Nuutila K, Laukkanen A, Lindford A, Juteau S, Nuopponen M, Vuola J, et al. Inhibition of

Skin Wound Contraction by Nanofibrillar Cellulose Hydrogel. Plast Reconstr Surg 2018;141. https://doi.org/10.1097/PRS.0000000000004168.

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Claims (22)

1. A nanofibrillar cellulose composition comprising nanofibrillar cellulose (NFC), cellulase and one or more bioactive agent(s), wherein the one or more bioactive agent(s) is(are) selected from vascular endothelial growth factors (VEGFs) such as VEGF-A, VEGF-C and VEGF-D; nerve growth factor (NGF); NGF-beta; platelet- derived growth factor (PDGF); fibroblast growth factors (FGFs) such as FGF1, FGF2, FGF3, FGF4, FGF5, FGF6, FGF7, FGF8, FGF9, FGF10, FGF11, FGF12, FGF13, FGF14, FGF15, FGF16, FGF17, FGF18, FGF19, FGF20, FGF21, FGF22 and FGF23; epidermal growth factor (EGF); tumor necrosis factor (TNF); transforming growth factors (TGFs) such as TGF-alpha, TGF-beta 1, TGF-beta 2 and TGF-beta 3; insulin- like growth factors (IGFs) such as IGF-I, IGF-II and des(l-3)-lGF-I (brain IGF-I); neurotrophin-3 (NT-3); brain-derived neurotrophic factor (BDNF); interleukins (ILs) such as IL-1, IL-1 alpha, IL-1 beta, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL- 11, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, IL-18, IL-19, IL-20, IL-21, IL-22, IL-23, IL- 24, IL-25, IL-26, IL-27, 11-28, IL-29, IL-30, IL-31, IL-32, IL-33, IL-35 and IL-36; whole cells; extracellular vesicles; and platelet-rich plasma (PRP).

2. The nanofibrillar cellulose composition according to claim 1, wherein said composition comprises NFC at a concentration of 0.1 to 3.5 % (w/w), preferably from 0.2 to 3.5 % (w/w).

3. The nanofibrillar cellulose composition according to claim 1 or 2, wherein said composition comprises one or more cellulase(s) at a concentration of 10 ug to 5000 Hg cellulase per mg of cellulose, preferably 50 ug to 3000 ug cellulase per mg of cellulose. N

< 4. The nanofibrillar cellulose composition according to any one of the preceding claims, 2 25 wherein the NFC in the composition is in a form of a hydrogel.

N 5. The nanofibrillar cellulose composition according to any one of the preceding claims, E wherein said NFC comprises cellulose nanofibrils and/or nanofibril bundles having N number average diameter between 1 and 200 nm, preferably between 2 and 100 nm. ©

S 6. The nanofibrillar cellulose composition according to any one of the preceding claims, O 30 wherein said NFC comprises native NFC, medical grade NFC and/or modified NFC, preferably medical grade NFC.

7. The nanofibrillar cellulose composition according to any one of the preceding claims, wherein said NFC is plant-derived.

8. The nanofibrillar cellulose composition according to any one of the preceding claims, wherein said NFC has a storage modulus between 0.3 and 50 Pa, preferably between 1 and 40 Pa, at a concentration of 0.5 % (w/w) in water.

9. The nanofibrillar cellulose composition according to any one of the preceding claims, wherein said NFC has yield stress between 1 and 50 Pa, preferably between 3 and 15 Pa, at a concentration of 0.5 % (w/w) in water.

10. The nanofibrillar cellulose composition according to any one of the preceding claims, wherein said NFC has zero-shear viscosity in the range of 1000-100000 Pa-s, such as in the range of 5000- 50000 Pa-s, at a concentration of 0.5 % (w/w) in water.

11. The nanofibrillar cellulose composition according to any one of the preceding claims, wherein said composition comprises one or more additional components selected from auxiliary agents and nutritional agents.

12. The nanofibrillar cellulose composition according to any one of the preceding claims, wherein said composition has a backing material attached thereto, or it is incorporated on a support.

13. The nanofibrillar cellulose composition according to any one of the preceding claims, wherein said composition is incorporated in a gauze or non-woven material.

14. A nanofibrillar cellulose composition comprising nanofibrillar cellulose, cellulase and e one or more bioactive agent(s), wherein the one or more bioactive agent(s) is(are) S selected from vascular endothelial growth factors (VEGFs) such as VEGF-A, VEGF- o C and VEGF-D; nerve growth factor (NGF); NGF-beta; platelet-derived growth factor O © (PDGF); fibroblast growth factors (FGFs) such as FGF1, FGF2, FGF3, FGF4, FGF5, N 25 FGF6, FGF7, FGF8, FGF9, FGF10, FGF11, FGF12, FGF13, FGF14, FGF15, FGF16, I T FGF17, FGF18, FGF19, FGF20, FGF21, FGF22 and FGF23; epidermal growth factor N (EGF); tumor necrosis factor (TNF); transforming growth factors (TGFs) such as TGF- 2 alpha, TGF-beta 1, TGF-beta 2 and TGF-beta 3; insulin-like growth factors (IGFs) O N such as IGF-I, IGF-II and des(l-3)-lGF-I (brain IGF-I); neurotrophin-3 (NT-3); brain- N 30 derived neurotrophic factor (BDNF); interleukins (ILs) such as IL-1, IL-1 alpha, IL-1 beta, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, IL-18, IL-19, IL-20, IL-21, IL-22, IL-23, IL-24, IL-25, IL-26, IL-27, 11-28, IL-

29, IL-30, IL-31, IL-32, IL-33, IL-35 and IL-36; whole cells; extracellular vesicles; and platelet-rich plasma (PRP), for use in wound treatment, optionally wherein the nanofibrillar cellulose composition is the nanofibrillar cellulose composition according to any one of claims 1 — 13.

15. Use of the nanofibrillar cellulose composition according to any one of claims 1 — 13 for manufacturing a wound treatment product.

16. A wound treatment product comprising the nanofibrillar cellulose composition according to any one of claims 1 — 13.

17. A method of producing a nanofibrillar cellulose composition, comprising: a) providing a component comprising cellulase b) providing i) a component comprising NFC and one or more bioactive agent(s), or ii) a component comprising NFC and a component comprising one or more bioactive agent(s); c) combining i) the component comprising cellulase and the component comprising NFC and one or more bioactive agent(s), or ii) the component comprising cellulase, the component comprising NFC and n the component comprising one or more bioactive agent(s); N O N 20 wherein the one or more bioactive agent(s) is(are) selected from vascular endothelial S growth factors (VEGFs) such as VEGF-A, VEGF-C and VEGF-D; nerve growth factor O (NGF); NGF-beta; platelet-derived growth factor (PDGF); fibroblast growth factors = (FGFs) such as FGF1, FGF2, FGF3, FGF4, FGF5, FGF6, FGF7, FGF8, FGF9, * FGF10, FGF11, FGF12, FGF13, FGF14, FGF15, FGF16, FGF17, FGF18, FGF19, N O 25 FGF20, FGF21, FGF22 and FGF23; epidermal growth factor (EGF); tumor necrosis 2 factor (TNF); transforming growth factors (TGFs) such as TGF-alpha, TGF-beta 1, S TGF-beta 2 and TGF-beta 3; insulin-like growth factors (IGFs) such as IGF-I, IGF-II and desf(l-3)-IGF-I (brain IGF-I); neurotrophin-3 (NT-3); brain-derived neurotrophic factor (BDNF); interleukins (ILs) such as IL-1, IL-1 alpha, IL-1 beta, IL-2, IL-3, IL-4, IL-

5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, IL-18, IL- 19, IL-20, IL-21, IL-22, IL-23, IL-24, IL-25, IL-26, IL-27, 11-28, IL-29, IL-30, IL-31, IL-32, IL-33, IL-35 and IL-36; whole cells; extracellular vesicles; and platelet-rich plasma (PRP).

18. The method according to claim 17, wherein the component of step b)i) or step b)ii) is in a form of a hydrogel.

19. The method according to claim 18, wherein the component of step b)i) or b)ii) is in freeze-dried form, optionally wherein the method further comprises: b2) reconstituting the freeze-dried component to form a hydrogel, wherein step b2) is performed between steps b) and c).

20. The method according to any one of claims 17 to 19, wherein the method further comprises: d) mixing the combined components of step c) to produce an essentially homogeneous distribution of components.

21. A kit comprising: a) a container comprising cellulase; and b) i) a container comprising NFC and one or more bioactive agent(s), or ii) a container comprising NFC and a container comprising one or more bioactive agent(s); wherein the one or more bioactive agent(s) is(are) selected from vascular endothelial growth factors (VEGFs) such as VEGF-A, VEGF-C and VEGF-D; nerve growth factor ™ (NGF); NGF-beta; platelet-derived growth factor (PDGF); fibroblast growth factors S (FGFs) such as FGF1, FGF2, FGF3, FGF4, FGF5, FGF6, FGF7, FGF8, FGF9, o FGF10, FGF11, FGF12, FGF13, FGF14, FGF15, FGF16, FGF17, FGF18, FGF19, = 25 FGF20, FGF21, FGF22 and FGF23; epidermal growth factor (EGF); tumor necrosis N factor (TNF); transforming growth factors (TGFs) such as TGF-alpha, TGF-beta 1, = TGF-beta 2 and TGF-beta 3; insulin-like growth factors (IGFs) such as IGF-I, IGF-II N and des(I-3)-IGF-I (brain IGF-I); neurotrophin-3 (NT-3); brain-derived neurotrophic 2 factor (BDNF); interleukins (ILs) such as IL-1, IL-1 alpha, IL-1 beta, IL-2, IL-3, IL-4, IL- & 30 5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, IL-18, IL- N 19, IL-20, IL-21, IL-22, IL-23, IL-24, IL-25, IL-26, IL-27, 11-28, IL-29, IL-30, IL-31, IL-32,

IL-33, IL-35 and IL-36; whole cells; extracellular vesicles; and platelet-rich plasma (PRP).

22. The kit according to claim 21, wherein the kit is for use in the method according to any one of claims 17 to 20. O N O N O S © N I a a N O O O O Al O N