The present invention relates to hydrogels based on polysaccharides, such as alginate and nanocellulose and particularly RGD conjugated alginate and RGD conjugated nanocellulose combined with fibrin for use as novel bioinks to be used with 3D Bioprinting technology and a combination of these novel bioinks with a coaxial printing needle. These novel bioinks are particularly suitable for 3D cell culturing of human fibroblasts and growing human skin. In this invention RGD-conjugated alginate is used in the formulation of the 3D Bioprinting bioink with non-conjugated alginate. The composition of the bioink is designed to provide optimal rheological properties which gives high printing fidelity. Nanocellulose is added to control rheological properties whereas fibrin is added to provide suitable environment for fibroblasts to proliferate and produce an extracellular matrix, preferably Collagen I. A critical aspect claimed by this invention is the presence of RGD peptide conjugated to alginate, which affects adhesion and spreading of human fibroblasts, as well as the presence of fibrin. The spreading of human fibroblasts activates the cells and results in upregulation of Collagen I production, which is a major component of the skin. Bioinks described herein were printed with and without a coaxial needle providing fast crosslinking upon bioprinting and giving optimal printing fidelity which resulted in high cell viability. Bioink described in this invention can be 3D bioprinted with or without human fibroblasts, but mixing and 3D bioprinting with human fibroblasts in the mode known as cell-laden hydrogel is preferred. Embodiments of this invention relate to human skin and particularly the dermis layer of the skin. Epidermis is the top layer of the skin and it consists of several types of cells such as keratinocytes, melanocytes and Langerhans cells. Keratinocytes are the most abundant cell type. Epidermis is much thinner than dermis which typically is 1-4 mm thick, depending on the location in the body. The invention describes how the bioink is mixed with cells, 3D bioprinted, and cultured to become a model for skin which can then be used for testing of cosmetics, skin care products and be used for transplantation. It can also be used for high throughput drug discovery, screening, and toxicity testing. Alternatively it can be directly implanted in a wound.
Skin is the human body's largest organ. It is composed of two layers; epidermis, which is the outermost layer and consists mainly of keratinocytes, which, during the process called stratification, are converted into dense layer(s) of keratin which act as a barrier. The second layer, dermis, is mainly composed of dermal fibroblasts which are responsible for production of extracellular matrix. The major component of extracellular matrix of dermis is Collagen I. During the human aging process, the production of Collagen I is decreased and also connections between the Collagen I network and fibroblasts decreases. This results not only in damage to the skin, but also the presence of wrinkles. The cosmetic and skin care industry has been working to develop products containing active substances which can enhance proliferation of fibroblasts and increase production of Collagen I. New products were evaluated in Europe until 2013 mostly by testing the skin products through animal testing. Since 2013, a ban has been levied against animal testing of cosmetic products in Europe and the cosmetic industry is researching to develop other models of human skin.
Additionally, 11 million people worldwide suffer from burn injuries requiring medical intervention. 300,000 people die every year due to burn injuries. The burns, which are second, third or fourth degree, require urgent treatments. Currently, skin grafts are used to help these patients. Autologous skin grafts are preferred but the burned patients often lack the undamaged skin to be transplanted. Patients' own skin, which could be grown in a laboratory, would help to save the lives of thousands of patients.
3D Bioprinting is an emerging technology which enables biofabrication of tissue and organs. The technology is based on using 3D bioprinters, which comprise a robotic arm that dispenses liquid biomaterial and cells in a pattern determined by CAD file blue prints to control the motion of the 3D bioprinter. It is taught herein that 3D Bioprinting technology may be used for biofabrication of human skin since the different layers can be printed with various cell densities with high resolution. The outcome of the 3D Bioprinting process will depend on the bioinks being used. Bioinks have the role of providing suitable rheological properties during 3D Bioprinting, cell viability, and also acting as scaffolds during tissue development.
Human fibroblasts need to attach in order to actively produce extracellular matrix. In native environments, such attachment takes place by binding to fibronectin, which contains Arg-Gly-Asp (RGD) domains that interact with cells through integrins, which are transmembrane cell adhesion receptors. Alginates have been used for many years as biomaterials for cell encapsulation and have many biomedical applications.
Cells do not bind to pure alginate. Conjugation of a variety of peptides facilitate and promote cell attachment. Peptide-coupled alginates can be prepared using aqueous carbodiimide chemistry as described by J. A. Rowley, G. Madlambayan, D. J. Mooney, Alginate hydrogels as synthetic extracellular matrix materials, Biomaterials 20 (1999), 45-53. Examples of materials described in this innovation are NOVATACH G/M RGD (GRGDSP-coupled high G or high M alginate), NOVATACH G VAPG (VAPG-coupled high G alginate), NOVATACH M REDV (REDV-coupled high M alginate) produced by FMC Biopolymers, NovaMatrix, Norway.
In this invention, a preparation of a new bioinks is described, such as bioinks composed of: RGD-modified alginate; fibrin with or without addition of nanocellulose or RGD-modified nanocellulose; and fibrin with addition of alginate. This invention also teaches using such bioinks for printing with human fibroblasts. RGD-modified alginate provides attachments sites for integrins at the surfaces of fibroblasts resulting in cell stretching. Cell stretching has been shown to upregulate production of Collagen I, which makes such 3D Bioprinted constructs preferable for use as a dermis model for testing active substances in cosmetics or skin care products, or for skin transplantation. This invention also describes using a coaxial needle to crosslink alginate during a 3D Bioprinting process. When dermis is developed the keratinocytes can be seeded or 3D Bioprinted on the top of such dermis layer while full skin is developing.
The accompanying drawings illustrate certain aspects of embodiments of the present invention, and should not be used to limit or define the invention. Together with the written description the drawings serve to explain certain principles of the invention.
a) Unmodified bioink b) RGD-modified Alginate c) RGD-modified Alginate and addition of TGFBeta to medium
The present invention has been described with reference to particular embodiments having various features. It will be apparent to those skilled in the art that various modifications and variations can be made in the practice of the present invention without departing from the scope or spirit of the invention. One skilled in the art will recognize that these features may be used singularly or in any combination based on the requirements and specifications of a given application or design. Embodiments comprising various features may also consist of or consist essentially of those various features. Other embodiments of the invention will be apparent to those skilled in the art from consideration of the specification and practice of the invention. The description of the invention provided is merely exemplary in nature and, thus, variations that do not depart from the essence of the invention are intended to be within the scope of the invention.
Before explaining at least one embodiment of the invention in detail, it is to be understood that the invention is not limited in its application to the details of construction and the arrangement of the components set forth in the following description or illustrated in the drawings. The invention is capable of other embodiments or of being practiced or carried out in various ways. Also, it is to be understood that the phraseology and terminology employed herein is for the purpose of description and should not be regarded as limiting.
Embodiments of the invention include RGD-modified alginate bioink products prepared by the methods described and include using the products in 3D Bioprinting operations.
a) Unmodified bioink b) RGD-modified Alginate c) RGD-modified Alginate and addition of TGFBeta to medium
To facilitate a better understanding of the present invention, the following examples of certain aspects of some embodiments are given. In no way should the following examples be read to limit, or define, the scope of the invention.
3D Bioprinting of Dermis-Like Model
Two different bioinks were prepared. The first bioink was composed of pure alginate with addition of nanocellulose to control rheological properties. The second bioink was prepared by combining RGD-modified alginate with nanocellulose to control rheological properties. Both bioinks had good printability. Six million primary human fibroblasts passage #3 were thawed and seeded into two 150 cm2 T-flasks. When the culture reached approximately 90% confluence, the cells were harvested using TrypLE and the flask was gently tapped to make the cells detach from the surface. The cells were counted (1.9 M cells/mL) with Tryphan-blue staining and the cell viability was calculated to ensure the cells were alive. The cells were then centrifuged and resuspended in medium and then seeded with 2,500 cells/cm2 into a T150 flask. The medium (DMEM, 1% GlutaMAX with 10% FBS and 1% Pen/Strep with phenol red) was changed three times per week. The cells were mixed with the bioinks to provide a final concentration of 5.2 million cells/ml and then carefully moved into the printer cartridge. Constructs were printed in a grid pattern in three layers with the dimensions of 6 mm×6 mm×1 mm (pressure: 24 kPa, feed rate: 10 mm/s) using the 3D-bioprinter INKREDIBLE from CELLINK AB, Sweden (see
Bioinks were prepared using aseptic techniques from fibrinogen powder purchased from Sigma and hydrogels of 3% nanocellulose and 2.6% alginate conjugated with GRGDSP-peptides acquired from FMC Biopolymers, NovaMatrix. The inks were made by mixing the components into homogeneous hydrogels. For the inks containing fibrinogen, the nanocellulose and alginate hydrogels were first mixed and the fibrinogen was dissolved with 200 μL/10 mg fibrinogen tris Buffered Saline (TBS) acquired from Fisher BioReagents. By using a SpeedMixer™ DAC 150.1 FV-K, the fibrinogen was mixed in the hydrogel to a homogeneous hydrogel composed of fibrinogen, nanocellulose and alginate. Different amounts of fibrinogen were added to hydrogel bioinks ranging from 10 mg to 500 mg per 1 ml bioink. Two different types of cells were used; primary adult human dermal fibroblasts (aHDFs) and primary human epidermal keratinocytes (HEKs) both acquired from LifeLine® Cell Technology. They were both cultured according to protocol from the supplier in cell specific culture medium (FibroLife® and DermaLife®, respectively) before mixing with bioinks and bioprinting. A thrombin solution was prepared with 10 units/ml thrombin in 100 mM CaCl2 to be able to crosslink the alginate and polymerize the fibrinogen simultaneously. The chosen construct model was a grid pattern in two layers. aHDFs were mixed in bionks in a concentration of 10 M cells/ml. Both lower and higher cell concentrations can be used. The printer used was a extrusion bioprinter (INKREDIBLE®, CELLINK®). The printing pressure for the fibrin based bioinks was between 12-23 kPa. After printing, the constructs were crosslinked and polymerized for 5 min using thrombin solution in 100 mM CaCl2 before placing in culture medium. The constructs were then cultured in FibroLife® medium for two weeks. After two weeks HEK cells were seeded (30 M/ml medium) and samples were incubated for another two weeks. The samples for analysis were taken at 7 and 14 days and 28 days. After constructs were sliced and stained for pro-collagen and Masson's trichrome staining to get visualization of collagen production within the constructs. There was positive effect of the addition of fibrin on cell morphology and production of Collagen I.
The constructs composed of fibroblasts laden RGD-alginate were prepared by 3D Bioprinting using a coaxial needle (see
One skilled in the art will recognize that the disclosed features may be used singularly, in any combination, or omitted based on the requirements and specifications of a given application or design. When an embodiment refers to “comprising” certain features, it is to be understood that the embodiments can alternatively “consist of” or “consist essentially of” any one or more of the features. Other embodiments of the invention will be apparent to those skilled in the art from consideration of the specification and practice of the invention.
It is noted in particular that where a range of values is provided in this specification, each value between the upper and lower limits of that range is also specifically disclosed. The upper and lower limits of these smaller ranges may independently be included or excluded in the range as well. The singular forms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise. It is intended that the specification and examples be considered as exemplary in nature and that variations that do not depart from the essence of the invention fall within the scope of the invention. Further, all of the references cited in this disclosure are each individually incorporated by reference herein in their entireties and as such are intended to provide an efficient way of supplementing the enabling disclosure of this invention as well as provide background detailing the level of ordinary skill in the art.
Filing Document | Filing Date | Country | Kind |
---|---|---|---|
PCT/US17/35861 | 6/3/2017 | WO | 00 |
Number | Date | Country | |
---|---|---|---|
62345597 | Jun 2016 | US |