WO2011155857A2 - The method of obtaining a tissue engineered product for the reconstruction and regeneration of bone tissue, the product and its use - Google Patents

Abstract

The subject matter of invention is a method for obtaining a tissue engineered product for the reconstruction and regeneration of bone tissue, the tissue engineered product, and its applications. In more detail: the invention represents the method for obtaining the tissue engineered product in cell culture conditions in vitro on degradable material. The product consists almost entirely of natural components, which are produced by cells seeded on a resorbable material. When the cells come from the donor who is also the recipient of the graft, the product is composed of his/her own biological material. Such composition ensures immunological compatibility, which does not occur in the case of biological material obtained from an incompatible donor or when the biological material is of animal origin, which is often the case in current clinical practice.

Description

The method of obtaining a tissue engineered product for the reconstruction and regeneration of bone tissue, the product and its use

The subject matter of invention is a method for obtaining a tissue engineered product for the reconstruction and regeneration of bone tissue, the tissue engineered product, and its applications. In more detail: the invention represents the method for obtaining the tissue engineered product by culturing cells on degradable resorbable material in vitro. The product consists almost entirely of natural components, which are produced by the cells seeded on the material. When the cells come from a donor who is also the recipient of the graft, the product is composed of his/her own biological material. The tissue-engineered material designed in such way may be used for minimally invasive treatment of maxillary sinus floor elevation, horizontal bone augmentation or as a filler in aesthetic medicine treatments. Such composition ensures immunological compatibility, which does not occur in the case of biological material obtained from an incompatible donor or when the biological material is of animal origin, which is often the case in current clinical practice.

An efficient regeneration of bone defects is a crucial clinical issue [1-4]. Bone defects arise due to osteolytic changes caused by inflammation, due to a surgical removal of tumors or a pathological regeneration of fractures. The need for bone regeneration applies to patients treated by means of tumor surgery, orthopaedics and laryngology. A controlled augmentation of bone is essential in reconstructive operations performed in maxillofacial surgery.

Not fully satisfying methods for bone regeneration or supporting the mechanical functions of bone are currently used in clinical practice. An autologous bone transplant is a golden standard [5-7]. It is a part of the patient's own bone, harvested for a healthy site, and placed into the defect. Such transplantation ensures a tissue regeneration which is neither linked with an immunological response toward the graft nor a risk for the transmission of infections [8]. Unfortunately, the method means a two-stage surgery in a given patient and a morbidity of the harvesting site [9, 10]. A technique used alternatively is reconstructing the bone defect by an allogenic graft, i.e. by a bone harvested from a donor, mostly a cadaver. [11]. Then, the risk of transmitting an infection from the donor to the recipient occurs, and bone regeneration in the defect cannot be guaranteed. Synthetic materials (ceramics, polymers or metals) are also commonly used as defect fillers and as growth factors placed on biomaterials used for bone regeneration [12]. It is then very important to pay attention to their proper biocompatibility, mechanical properties and integration with the healthy tissues. None of these materials meet all the specified requirements. Guided tissue regeneration (TGR) is a very broad discipline, covering many clinical procedures in order to re-create the non-produced or damaged anatomical structures. Treatments are performed on both bone and soft tissue.

The commonly used TGR treatments in oral surgery operations are lifting of the sinus floor in both the open and the closed method. The first type of surgery is a highly invasive method, while the other one has a much more limited impact. Modern tissue- engineered products are designed to limit the invasiveness of procedures through the transition from the open to the closed type. At the same time, it is expected that the percentage of successful treatments will increase, the amount of the regenerated bone will grow, and the time of the osteointegration of the implants will be shorter.

Another widely encountered regenerative surgery is a horizontal alveolar bone augmentation. Despite the use of restorative techniques described above, the percentage of failures in this group is significant. Recent trends suggest a shift away from operating the conventional techniques of wide open spaces in favour of the tunnel method anticipating a small cut in the area distant from the bone defect. Then, after a preparation of the tunnel between the cortical bone plate and the periosteum, the osteoinductive material is deposited in the previously generated lodge. This technique has difficulties, especially when using large particles of bone material. An optimal solution in such case seems to be the use of a flexible material, or even an amorphous one.

A frequently performed procedure in aesthetic medicine is to fill grooves and other defects of the subcutaneous tissue. Materials currently used for this purpose are hyaluronic acid derivatives. Unfortunately, they are characterized by a relatively low stability in the tissue after application. This is due to the resorption of the material caused by the host's defense cells. A tissue engineered product of an autogenous origin would be devoid of this defect. In addition, living and active cells, applied in the area of the tissue defect, would have the ability to proliferate and produce de novo collagen and other extracellular matrix components.

Tissue engineering, a new interdisciplinary field of regenerative medicine, has emerged in order to search for methods for tissue regeneration due to the unsatisfactory results of the conventionally used methods and a great clinical need. The aim of tissue engineering is to create biological substitutes to restore, maintain or augment the functions of tissues [13]. The tissue engineered products which could enable tissue regeneration may be viable, modified or non-modified cells, growth factors or cells placed in a scaffold. The name 'bone tissue engineered product' thus means every construct which consists of a carrier in which bone cells are placed or carriers which release bone stimulating growth factors [14]. Such products may be prepared using cell cultures in vitro which precede the implantation [15]. The products may also be prepared during surgical procedures (e.g. by mixing the implant with cells isolated from bone marrow [16] or with a platelet-rich plasma [17, 18]. The products may be created in the recipient's body, as in an„incubator", before they are implanted to the defect site [19, 20]. Bone tissue engineering pays special attention to the proper mechanical properties of the graft. The majority of products which use the available biomaterials, i.e. ceramics and polymers, are implanted to non-load- bearing bone sites, due to their bnttleness. Tissue engineered product (TEP) is a legal concept/has a legal definition which was introduced in the resolution of the European Council No.1394/2007 (Products for advanced therapy), regulating the marketing rules for such products.

The viable part of the graft consists of cells which usually come from the patient's own body. The task for the cells within a scaffold is to create tissue and factors which would stimulate the neighboring tissues to take part in the regeneration of the defect site. Proper cell attachment to the scaffold should be guaranteed in order to obtain a differentiated bone cell phenotype. The bone cells which are fully active synthesize the extracellular matrix proteins characteristic for bone tissue, mainly collagen type I. The Precursor cells derived from bone tissue [21], mesenchymal stem cells from bone marrow [22], adipose tissue [23] or umbilical cord and fetal membranes [24, 25]— are used in experimental settings.

The scaffold made from synthetic of natural materials guarantees a three- dimensional growth of the tissue. Ceramics, polymers or their composites are mostly taken into account in bone tissue engineering. Great attention is paid to degradable materials, which vanish a certain time after the implantation [26]. Degradable polyesters, such as polylactates or polyglycolides, are approved to be used clinically as implanted materials (as sutures or screws) [27]. Therefore, they are widely investigated as scaffolds for bone tissue engineering [28]. Polylactide (PLA), poly-L-lactide or their copolymers with glycolide acid are the most widely used scaffolds in tissue engineering. [29, 30]. The main advantage of their use is that the products of their degradation (lactate and glycolide) are metabolized (resorbed) via natural metabolic pathways [31]. The less advantageous features of the materials are their low mechanical strength and poor adherence of the osteoblatic cells to their surfaces.

A lot of research is performed to optimize polymer scaffolds regarding their composition and architecture [32]. Different compositions of polylactate and polyglycolide, as well as adding fillers, such as inorganic molecules: calcium phosphates/carbonates or silica combinations - positively influence the materials' characteristics for tissue engineering [33-36]. Such modifications enhance cell differentiation towards fully active osteoblastic cells [37-39]. Modifications with collagen [40, 41], amine groups [42] or producing surfaces of an improved protein affinity [43] lead to better cell adhesion. The mechanical strength may be augmented by adding nanoparticles (eg. carbon nanotubes [44]).

It has already been shown that the architecture of the scaffold is crucial for bone cells placed within it. Fibrous structure stimulates cell proliferation and differentiation towards mature bone cells, due to its resemblance of the structure of fibrous elements of the native bone tissue. It was proved that a nanosized structure of the fibers enhances the ostoblast differentiation and tissue mineralization [45], and that the arrangement and the diameter of the fibers influence the osteoblast adhesion, proliferation and differentiation [46]. Electrospinning, a method for obtaining polymer fibers and their modifications, has been elaborated. Therefore, it is possible to produce three-dimensional fibrous scaffolds for obtaining a variety of fibers and pore dimensions [47, 48].

A proper sterilization process is important for the practical use of the polymers as implantable materials. Each type of sterilization influences the scaffold shape, the polymer molecular weight, and thus also the biocompatibility of the scaffold [49-51].

The optimization of the materials described above, i.e. of their composition, architecture and sterilization process, additionally affects the manner and speed of scaffold degradation. [35, 52-56]. The degradation rate of an implanted synthetic material is frequently unpredictable, but very important from the practical point of view. An excess of the degradation by-products (lactic and glycolic acids) decreases the viability of the implanted cells and is toxic for the neighboring tissues, due to the pH drop in the implant vicinity [57-59]. For example, in 6% of patients with polyester implanted into bone, a foreign body reaction occurred [57]. Nevertheless, the classical schema of using polymers in tissue engineering comprises polyester degradation after the introducing into the patient's body.

Hutmacher, in his review from 2000, distinguished two possible strategies to obtain a tissue engineered product on a degradable material [60]. According to the first strategy, the scaffold constitutes the main component of the tissue substitute, regarding its shape and mechanical properties during cell seeding in in vitro conditions up to the time of the replacement of the substitute by patient's own tissue. According to the other strategy, the tissue produced by the cells in vitro replaces the degradable scaffold. Then, the tissue, built from the extracellular matrix proteins produced by the cells, is capable of fulfilling the mechanical function of a graft. The references cited in the review with regard to the latter strategy in tissue engineering, describe studies which actually do not employ this strategy.

The prolonged cell cultures on degradable scaffolds have to date been performed by tissue engineering research teams working on blood vessels and valves [61, 62], cartilage [63, 64] and bone regeneration [65, 66]. It was shown that a 70% degradation of the scaffold prepared from of collagen and glycosaminoglycans accompanied an enhanced differentiation of the osteoblastic cell line (hFOB) after 5 weeks of an in vitro culture [65]. Cartilaginous tissue was obtained on a scaffold of polyglycolide, whose mechanical properties reached 40% of the native tissue after 12 weeks of an in vitro cell culture [63].

Stimulation of cells to produce tissue in vitro is inevitable for obtaining a tissue engineered product in a prolonged culture on degradable material. The main component of the extracellular matrix of bone is collagen type I. The stimulation of collagen type I synthesis is reached by adding biochemical agents to cells, e.g. ascorbic acid [67, 68], growth factors in the medium or released gradually from the scaffolds [65, 69], or by a mechanical stimulation of the cells [70]. Lactic acid, produced physiologically in hypoxic tissues, is a strong stimulant of collagen synthesis and maturation [71, 72]. The effect was already confirmed in vitro in fibroblast cell cultures [73-75]. The synthesis of collagen type I was enhanced by 70% in comparison to the control in fibroblast cell cultures after adding lactic acid to the culture medium [76].

The influence of lactate, added to the culture medium or released by resorbable scaffolds, on the production of collagen type I by osteoblastic cells, has not been investigated so far. It was only shown that lactate added to an osteoblast culture inhibited the synthesis of the vascular-endothelial growth factor (VEGF) [77], as opposed to the results obtained for cultures of other cell types: the endothelium [78] and macrophages [79].

In the patent application WO2009076594 (published: 2009-06-18) bone/collagen composites and their applications are described. The composite comprises bone and collagen, wherein the collagen has been acid treated, and cross-linked via dehydrothermal treatment or by a cross-linking agent (e.g. a citric acid derivative) under a compressive force of at least approximately 40MPa.

The patent applications US20040037813 (published: 2004-02-26) and US20080038352 (published: 2008-02-14) describe the formation and use of the electroprocessed collagen, including its use as extracellular matrix and, together with the cells, its use in forming the engineered tissue. The engineered tissue may include the synthetic manufacture of specific organs or tissues which may be implanted into a recipient. The electroprocessed collagen may also be combined with other molecules in order to deliver substances to the site of the application or implantation of the electroprocessed collagen. The collagen or collagen/cell suspension is electrodeposited onto a substrate to form tissues and organs.

The patent description of US6730252 (published: 2004-05-04) presents the use of Fused Deposition Modeling to construct three-dimensional (3D) bioresorbable scaffolds from bioresorbable polymers, such as polycaprolactone (PCL), or from composites of bioresorbable polymers and ceramics, such as polycaprolactone/hydroxyapatite (PCI/HA). The incorporation of a bioresorbable ceramic to produce a hybrid/composite material support provides the desired degradation and resorption kinetics. Such composite material improves the biocompatibility and hard tissue integration, and allows for an increased initial flash spread of the serum proteins. The basic resorption products of the composite also avoids the formation of an unfavorable environment for hard tissue cells due to a decreased pH. The scaffolds have their applications in tissue engineering, e.g. in tissue engineering of bone and cartilage.

The patent applications WO2006106506 (published: 2006-10-12) and US20090074832 (published: 2009-03-19) describe the manufacturing of electrospun elements possessing continuous or gradual changes of the gradients of porosity, average pore size, weight per volume and/or of agents for promoting cell colonization, differentiation, extravasation and/or migration. Methods of manufacturing and using the product in tissue regeneration are also presented. In the patent application P-385197 (published: 2009-11-23) a method for obtaining a tissue engineered product and the product for the reconstruction and regeneration of bone tissue are described. The method is based on producing a three-dimensional scaffold of synthetic calcium carbonate and culturing osteoblastic cells in dynamic culture conditions. The admixtures do not constitute more than 10%, the scaffold possesses an opened porosity (40-90% of porosity in total), the interconnected pores' dimensions are from 100 to 500μπι. Preferably, the osteogenic cells are isolated from the donor who is the recipient of the graft.

Despite the described inventions, there is a need for a method for obtaining a tissue engineered product of a highly advanced degradation of the polymer scaffold reached already in vitro, i.e. before the implantation. It is a crucial advantage over the situation when the degradation of the material occurs in vivo, only after the implantation, because the degradation rate is then unpredictable. The uncontrolled local increase of the degradation by-product (lactate or glycolide) concentration decreases the viability of the implanted cells and is toxic for the neighboring tissues, due to the drop of pH in the graft vicinity. A prolonged inflammation induced by the aciditation may lead to the improper functioning of the substitute and graft loss. In the presented invention the material degradation is used to stimulate tissue growth and is fully controllable in the described experimental settings.

The aim of the described invention is to limit the use of synthetic materials only to the in vitro phase of obtaining the products, and therefore prevent side effects caused by the scaffold degradation at the implantation site. Although the product is produced in vitro, it possesses the characteristics of an autologous graft, which is the golden standard in transplantology.

The described invention realizes the presented aim and overcomes the problems of the current limitations of technology. The invention presents a method of obtaining a tissue engineered product by the means of a cell culture in vitro on a resorbable scaffold. The product consists almost entirely of natural components, which are produced by cells seeded on the material. When the cells come from the donor who is also the recipient of the graft, the product is composed of his/her own biological material. Such composition ensures immunological compatibility, which does not occur in the case of biological material obtained from an incompatible donor or when the biological material is of animal origin - which is often the case in current clinical practice. The subject of invention is a method for obtaining a tissue engineered product for the reconstruction and regeneration of bone tissue, prepared by culturing viable cells of an osteogenic potential or other cells producing collagen type I on a biodegradable scaffold of a complex structure, characteristic in that the product is ready for implantation after reaching an advanced scaffold degradation, accompanied by an advanced production of the extracellular matrix proteins in vitro, by the cells seeded on the scaffold, prepared from a mixture of polymers/copolymers, or polymers/copolymers modified with fillers, while cells are isolated from the donor's tissue and then cultured in a proper medium for each cell type in vitro, until they reach an appropriate number, preferably 70-80% of confluence in culture; then, the cells are detached from the culture surface, resuspended in the proper medium, and counted, whereas the scaffolds immersed in culture medium are located in a device with a constant flow of the culture medium, or they are located in the culture dishes; then, the suspension of cells in proper concentration is put into the device or into the wells of the culture dish; then, they are located in an incubator of a constant humidity above 95%, temperature of 37°C and the presence of 5% carbon dioxide; then, the culture lasts for a minimum of 7 days, while the culture medium of the same composition is exchanged at least once; then, the products are replaced into the culture dish and cultured further until an advanced degradation of the polymeric material is reached, i.e., when more than 50% of lactic acid is released from the material - after that, the product is ready for application. Preferably, the substrate is a material releasing lactic acid, particularly polymers and copolymers of the lactic acid, preferably selected from the PLA, D-PLA, L-PLA, LD-PLA, and block copolymers, grafted or statistical, of the above varieties of the PLA with each other and with the PGA, PEG (poly (ethylene glycol)), and PCL, while the lactic acid is released by the substrate in the environment of the culture medium and stimulates osteogenic cells, and other cells capable of the secretion of type I collagen, embedded in the substrate, for the production of collagen type I, which is an essential component of the extracellular matrix.

Preferably, as a substrate for the cells are used materials releasing lactic acid and/or modified materials, preferably different varieties of the PLA and its copolymers, preferably selected from the PLA, D-PLA, L-PLA, LD-PLA, and block copolymers, grafted or statistical of the above varieties of the PLA with each other and with the PGA, PEG, and PCL, with fillers preferably in the form of dispersed powder of crystalline or amorphous salts of lactic acid, preferably selected from calcium lactate, sodium lactate, potassium lactate, and magnesium lactate, being an additional source of lactic acid. Preferably, as a substrate for the cells are used materials releasing lactic acid and/or modified materials, preferably different varieties of PLA and its copolymers, preferably selected from PLA, D-PLA, L-PLA, LD-PLA, and block copolymers, grafted or statistical, of these varieties of PLA with each other and with the PGA, PEG (poly (ethylene glycol)) and PCL, preferably containing ceramic fillers in the form of nanoparticles or microparticles in form of powder or ceramic granules, particularly stoichiometric and nonstoichiometric calcium phosphates, calcium carbonates, silicon oxides, and multiphase materials from these groups.

Preferably, as a substrate for the cells are used materials releasing lactic acid and/or materials with modifications in the form of additives releasing growth factors, while the polymers and copolymers are preferably with a filler in the form of powder or granules of a demineralized bone matrix.

Preferably, the embedded cells are cells of an osteoblastic potential, in particular cells isolated from bone tissue or progenitor cells, including those isolated from adipose tissue, bone marrow, peripheral blood, foetal tissues or cells of an angiogenic potential, in particular cells isolated from the endothelium, progenitor cells or cells of an osteoclastic phenotype.

Preferably, the cells embedded in one scaffold may be cells of a different phenotype and a different potential.

Preferably, the seeded cells are preferably cells isolated for mammalian tissues, preferably human, demonstrating species compatibility, and in case of application in human preferably as autologous graft.

Preferably, the cells building the graft are any cells able to synthesize the extracellular matrix characteristic for connective tissue.

Preferably, collagen fibers are formed, which are the base for the proliferation of cells, whose activity intensifies the scaffold degradation, causing a further release of lactic acid and, due to the presence of the lactic acid, a further stimulation of collagen type I and extracellular matrix contribution in the product, which causes a substitution of the original scaffold with tissue obtained in in vitro conditions before introducing into the recipient's body.

Preferably, the lactic acid released by the lactate-based scaffold stimulates and controls the rate of collagen fiber formation, and the scaffold shape determines the three-dimensional fiber architecture of the collagen produced by the seeded cells in the cell culture. Preferably, the lactic acid concentration does not exceed the toxic concentration for the given cells.

The next subject of invention is a tissue engineered product for the reconstruction and regeneration of bone tissue, obtained using the method according to the above, prepared by culturing viable cells of an osteogenic potential or other cells producing collagen type I on a biodegradable scaffold of a complex structure, characterized in that the product is ready for implantation after reaching an advanced scaffold degradation, accompanied by an advanced production of extracellular matrix proteins in vitro, by cells seeded on the scaffold.

Preferably, the substrate is a material releasing lactic acid and/or modified material, preferably particularly polymers and copolymers of the lactic acid, preferably selected from the PLA, D-PLA, L-PLA, LD-PLA, and block copolymers, grafted or statistical, of the above varieties of the PLA with each other and with the PGA, PEG, and PCL, while the lactic acid is released by the substrate in the environment of the culture medium and stimulates the osteogenic cells and other cells capable of the secretion of type I collagen, embedded in the substrate for producing collagen type I, which is the basic component of the extracellular matrix.

Preferably, as a substrate for the cells is used a material releasing lactic acid and/or modified material, preferably different varieties of the PLA and its copolymers, preferably selected from the PLA, D-PLA, L-PLA, LD-PLA, and block copolymers, grafted or statistical, of the above varieties of PLA with each other and with the PGA, PEG and PCL, with fillers preferably in the form of a dispersed powder of crystalline or amorphous salts of the lactic acid, preferably selected from calcium lactate, sodium lactate, potassium lactate, and magnesium lactate, being an additional source of lactic acid.

Preferably, the substrate for the cells is a material releasing lactic acid and/or modified material, preferably different varieties of the PLA and its copolymers, preferably selected from the PLA, D-PLA, L-PLA, LD-PLA, and block copolymers, grafted or statistical, of the above varieties of the PLA with each other and with the PGA, PEG (poly (ethylene glycol)) and PCL, preferably containing ceramic fillers in the form of nanoparticles or microparticles in the form of powder or ceramic granules, particularly stoichiometric and nonstoichiometric calcium phosphates, calcium carbonates, silicon oxides, and multiphase materials of these groups.

Preferably, the embedded cells are cells of an osteoblastic potential, in particular cells isolated from bone tissue or progenitor cells, including those isolated from adipose tissue, bone marrow, peripheral blood, foetal tissues or cells of an angiogenic potential, in particular cells isolated from the endothelium, progenitor cells or cells of an osteoclastic phenotype.

Preferably, the cells embedded in one scaffold may be cells of a different phenotype and a different potential.

Preferably, the seeded cells are preferably cells isolated from mammalian tissues, preferably human, demonstrating species compatibility, and, in the case of its application in a human, preferably as autologous graft.

Preferably, the cells building the graft are any cells able to synthesize the extracellular matrix characteristic for connective tissue.

Preferably, collagen fibers are formed, which are the base for the proliferation of cells, whose activity intensifies the scaffold degradation, causing a further release of lactic acid, and, due to the presence of the lactic acid, a further stimulation of collagen type I and extracellular matrix contribution in the product, which causes a substitution of the original scaffold with tissue obtained in in vitro conditions before the implantation into the recipient's body.

Preferably, the lactic acid released by the lactate-based scaffold stimulates and controls the rate of collagen fiber formation, and the scaffold shape determines the three-dimensional fiber architecture of the collagen produced by the seeded cells in cell culture.

Preferably, the lactic acid concentration does not exceed the toxic concentration for the given cells.

Preferably, the product consists of a n inimum of 80% natural components produced by the cells seeded on the scaffold.

Preferably, the cells come from the donor who is the recipient of the graft, which is composed only of the patient's own biological material so that immunological compatibility is provided.

Preferably, the product possesses autologous graft features.

The next subject of the invention is the use of the tissue engineered product described above, in which the product is implanted after reaching advanced scaffold degradation accompanied by an advanced production of extracellular matrix proteins in vitro, to be implanted in bone tissue or soft tissue defects, depending on type of cells used and/or to create specific tissue.

Preferably, the product is applied in the regeneration of bone tissue in maxillary sinus floor elevation. Preferably, the product is used to produce bone after subperiosteal application.

Preferably, the product is applied in the regeneration of soft tissue in the procedures procedures in aesthetic medicine.

Figures attached provide a better explanation of the invention.

Figure 1 presents a scaffold before the cell culture (scanning electron microscope),

Figure 2 presents a scaffold with tissue after 2 weeks of cell culture in dynamic conditions (scanning electron microscope),

Figure 3 presents an 8-week osteoblastic cell culture on a scaffold modified with silica microparticles.

Figure 4 presents an 8-week osteoblastic cell culture on a scaffold modified with silica nanoparticles.

Figure 5 presents explants of the tissue engineered products after a-4 week in vivo implantation.

Figure 6 presents connective tissue with blood vessels, which fills a graft obtained from a polyester scaffold modified by silica nanoparticles (hematoxylin and eosin staining).

Figure 7 presents connective tissue with blood vessels, which fills a graft obtained from a polyester scaffold modified by silica microparticles (hematoxylin and eosin stainig).

Figure 8 presents collagen which fills the explant of a polyester scaffold (Sirius red staining in a polarized light).

Figure 9 presents the lactate concentration in the culture medium in cell cultures on a degradable scaffold.

Figure 10 presents the number of the osteoblasts on polyester scaffolds at several timepoints of the culture.

Figure 11 presents the concentration of osteocalcin in the cell cultures on a scaffold on Day 14. and 21. of culture (the result is normalized to the cell number).

Figura 12 presents the amount of collagen in the cell cultures on scaffolds at several timepoints.

Figure 13 presents spaces between the scaffold fibers filled with a tissue-like structure (scanning electron microscope),

Figure 14 presents the concentration of collagen type I in a 21 -day culture in different lactate concentrations in the culture medium (the result is normalized to the cell number). Figure 15 presents the concentration of collagen type I in osteoblastic cultures with and without the addition of lactic acid to the medium. Figure 16 presents the expression of collagen type I gene in an osteoblastic cell culture with and without the addition of lactic acid to the medium.

Figure 17 presents the concentration of collagen type I in fibroblast cultures at several timepoints.

Figure 18 presents the concentration of collagen type I in fibroblast cultures with lactic acid and ascorbic acid, or with lactic acid only - added to the culture medium.

Examples:

Example 1. General method for obtaining the product.

A scaffold is produced from a mixture of proper polymers/copolymers or polymers/copolymers modified with fillers. The prepared scaffold is sterilized by radiation (25kGy dose) and kept in sterile conditions until the preparation of the product. Cells are isolated from the donor's tissue (e.g. from bone, bone marrow, peripheral blood, blood vessels, connective tissue). The cells are then cultured in the culture medium proper for each cell type (e.g. for cells derived from bone tissue, the medium consists of DMEM (GIBCO BRL catalog num.22320) enriched with inactivated fetal calf serum at 10% concentration, with the addition of an antibiotic in the form of Antibiotic-Antimycotic (GIBCO, catalog num. 15240096, consisting of 10.000 U of penicillin in the form of natrium salt of penicillin G, 10.000 μg streptomycin in the form of streptomycin sulphate) and 25 g amphotericin B/ml - in the form of Fungizone® of 0.85% in saline) at the concentration of 1%, L-glutamine at the concentration of 2mM (GIBCO BRL) and vitamin C at the concentration of 120 μΜ in the form of L-ascorbic acid 2-phosphate (SIGMA)). The culture of isolated cells is performed in culture dishes in an incubator of a constant humidity above 95%, temperature of 37°C and the presence of 5% carbon dioxide until they reach the appropriate number, preferably 70-80% of confluence in the culture. Then, the cells are detached from the culture surface by coUagenase and trypsin solutions, and are resuspended in the proper medium, and counted. The scaffolds immersed in the culture medium are located in the basket of a device with a constant flow of the culture medium or are located in the culture dishes; then, the suspension of the cells is added in a proper concentration (e.g. 0.5 million cells per scaffold 5x5x5mm). The setting is located in an incubator of a constant humidity above 95%, temperature of 37°C and the presence of 5% carbon dioxide. The device with the constant flow of the culture medium is then placed on a magnetic stirrer, and the velocity of the stir is kept at about 60rpm. When the device is used, the culture lasts for 14 days, but after 7 days, the culture medium is exchanged - for the same composition. The products are then replaced into the culture dish and cultured further until an advanced degradation of the scaffold occurs (when 80% of the lactic acid is released from the scaffold). In the case of culture on culture dishes only, the products are kept in the described conditions until an advanced degradation of the scaffold occurs (when 80% of the lactic acid is released from the scaffold). The culture medium is changed 2 times a week while preparing the product. After that phase the product is ready to be used. The degree of the scaffold degradation in the product is assessed by the method described in example 2. Additionally, an assessment of the tissue quality is performed (e.g. depending on the type of the tissue: the number of the cells (PicoGreen), the cell differentiation (osteocalcin concentration, alkaline phosphatase activity), gene expression (real-time PCR), the amount of the extracellular matrix (measurement of the hydroxyproline content, collagen type I content), the arrangement of the extracellular matrix (microscopic observation).

Example 2. Determination of the lactic acid content.

Determination of the lactic acid content enables us to determine the degree of the degradation of the bone scaffold.

The method of determination of the bone scaffold degradation degree:

The sample of the bone scaffold of a known initial weight, fresh or after cell culturing, is rinsed twice, in a 10-fold excess of distilled water, for 5 minutes each time. The residue insoluble in water is drained and dried at 40°C for 24 hours. The completely dried sample is weighed carefully, and then sulfuric acid is added at a concentration of 20 wt%, of a mass 5 times greater than the mass of the sample after drying. It is allowed to complete the hydrolysis at 40°C for 24 hours. Then, the lactic acid content can be determined in the obtained sample, according to one of the known methods:

A. Lactic acid is distilled under vacuum (120°C, 1.5 kPa), and then the distillate is titrated with a standard sodium hydroxide solution, using phenolphthalein as an indicator.

B. The sample is filtered and neutralized to pH 7, diluted according to the given method, and the concentration of the lactate ions is estimated using HPLC.

C. The sample is neutralized to pH 7, diluted according to the given method, and the lactate ion concentration is estimated by a suitable electrode, such as EDGE.

The degradation degree of the bone scaffold is equal to the mass of the sample after the culture, divided by the mass of the initial bone scaffold sample (without cells). Example 3. Method of preparing a tissue engineering product during a long-term in vitro culture of osteogenic cells on the scaffolds from polyester materials (PLLA- PLGA unmodified and modified with silica in the form of nanoparticles or microparticles).

A mixture of PLGA and PLLA (55mg) is dissolved in 1,4-dioxane, then, adding

270mg of porogen ( aCl) particles with a diameter of 250-500 microns per sample. The mixture is then frozen in liquid nitrogen and freeze-dried for a rninimum of 10 days. Then, the samples (325mg each) are compressed in cylindrical moulds under a pressure of 8 MPa at r.t. Then, NaCl is washed off by an immersion in distilled water for at least 3 days, changing the water twice a day. After this time, the scaffold samples are dried for 24 hours in the air, and then under vacuum for the next 8 hours. The scaffolds, modified with silica, are obtained by adding nanosilica particles, with a diameter of ca. 10 nm, or microsilica particles, with grain sizes of ca. 10 microns, to the starting mixture of the polymers and the porogen.

The product, which is sterile (due to the radiation sterilization with a dose of

25kGy), in the form of a cylinder is deareated in sterile conditions. The scaffold immersed in the culture medium (of the composition described below) is kept for 48h at the temp, of 37 °C in order to moisten the surface. Fresh culture medium is added before the cell culture.

The cells are isolated from small pieces of the donor's bone tissue (explants) through mechanical purification, in order to remove all tissues except bone, and are cut into pieces ab. the size of 1mm. The bone pieces are then digested in collagenase of 37°C overnight. The tissues which were digested are then removed, whereas the bone pieces are washed in PBS, and then located in culture bottlers in a culture medium of the following composition: DMEM (GIBCO BRL catalog no.22320) enriched with inactivated fetal calf serum of 10% concentration, with the addition of an antibiotic in the form of Antibiotic- Antimycotic (GIBCO, catalog no.15240096, consisting of 10.000 U of penicillin in the form of natrium salt of penicillin G, 10.000 μg of streptomycin in the form of streptomycin sulphate), and 25μg of amphotericin B/ml - in the form of Fungizone® of 0.85% in saline) at the concentration of 1%, L-glutarnine at the concentration of 2mM (GIBCO BRL), and vitamin C at the concentration of 120 μΜ in the form of L-ascorbic acid 2-phosphate (SIGMA)). The procedure is performed in sterile conditions. The culture is performed in an incubator of a constant humidity above 95%, temperature of 37°C and the presence of 5% carbon dioxide. After reaching confluence, the cells are detached from the surface by trypsin digestion; then, the cells are centrifuged, counted, and resuspended in the mentioned culture medium. The scaffolds are placed in a basket of a device with a constant flow of the culture medium or are located in the culture dishes; then, the suspension of the cells is added to the culture medium (in a large volume of the medium, e.g. 500ml) at the concentration of 300.000 cells per scaffold. There is only one type of scaffold per device (e.g. modified or non-modified PLGA scaffolds). The setting is then located in an incubator of a constant humidity above 95%, temperature of 37°C, and the presence of 5% carbon dioxide. The culture lasts for 14 days, but after 7 days the culture medium is exchanged - for the same composition. At that time the viability of the cells on the scaffold is measured on a randomly selected scaffold. The culture lasts further, for 14 days. After that, the product is obtained: it consists of the degraded scaffold filled with viable cells and the extracellular matrix produced by the cells (Fig.l and 2). (The description of the methods and results of implanting such products into the experimental animal tissues are provided in example 4).

The products are then replaced from the device into a sterile culture dish. The medium is changed 2 times a week. During the culture, scaffold degradation and tissue growth are observed macroscopically and microscopically. The product of a total disintegration of the scaffold is obtained after 8 weeks of culture, which is preceded by a 2- week culture in the conditions of a constant flow of the medium. The pieces of the scaffold are linked with a compact tissue structure (Fig. 3 and 4). The advanced degradation of the scaffold is confirmed by the size exclusion chromatography (SEC).

Example 4. Assessment of the products described in examples 1 and 3 after the implantation into the tissues of experimental animals.

The obtained scaffolds of a highly-advanced degradation are implanted into the tissues of experimental animals (the description of obtaining the product is provided in examples 1 and 3). Animals without cellular immune defense (SCID mice) were chosen, because the products consist human viable cells. A cut is done on a animal's back, and the tissues are separated to place the product. The control implant (without cells), kept for 48h before implantation in culture medium in sterile conditions, is implanted on one side of the animal's body, while the tissue engineered product is placed on the other side.

The implantation lasts for 4 or 13 weeks. Several quantitative and qualitative assesment methods are used to compare the experimental and control implants. The grafts prepared from polyster scaffolds, modified with microsilica particels, both experimental and control, are compact, robust, vascularised, and integrated with the animal tissues after a 4-week subcutaneous implantation (Fig. 5). There is no difference in the dimensions of the control and experimental grafts.

After a 13 -week implantation, the materials seeded with cells are degraded to a more advanced degree than the control implants. The cells caused faster degradation of the scaffold, which is observed in the form of a smaller volume of the explant, and in a quantitative assessment of the polymer chain changes, by size exclusion chromatography.

The products based on a polyester scaffold modified with silica in the form of nano- sized particles are also compact, while the experimental and control explants are macroscopically indistinguishable. There is no fibrous capsule around the implants, nor macroscopic signs of inflammation after both implantation periods.

A robust vascularised connective tissue is observed within the scaffold pores after the in vivo culture for both types of biomaterials (Fig 6 and 7). Collagen fibers shining in polarized light are visualized by Sirius red staining (Fig. 8).

Histological sections are prepared to confirm the presence of tissue produced by cells seeded on the scaffold. In the case of the non-modified polyester scaffolds, after 13 weeks in vivo, immunochistochemical dyes of frozen slices are additionally performed. Specific antibodies against the human collagen type I, human osteopontin and human osteocalcin are used. Positive stainings of above listed proteins are obtained in experimental implants, which proves the viability of the human cells and their ability for human extracellular matrix synthesis after implantation in vivo.

Example 5. Prolonged in vitro culture of osteogenic cells on polyester (PLA) scaffold.

Samples are produced by electrospinning in the following way: First, the polymer solution in chloroform is prepared in a glass jar with a magnetic stirrer. The clear solution is placed in the syringe, and the syringe is placed in the syringe pump. The syringe is connected to a metal nozzle with a plastic pipe. The nozzle is connected to a high voltage power supply. The polymer solution supplied by the syringe pump to the nozzle is getting electrical charges. After leaving the nozzle, the electrical charges are repelled by the coulomb forces from the nozzle, and the solution is accelerated in the electric field formed between the nozzle and the surroundings (earthed collecting electrode). This causes the elongation of the polymer solution filament and formation of thin fibers. Due to the solvent evaporation, the fibers solidify and are collected on the collecting electrode, which is connected to the ground potential.

The obtained scaffolds are immersed in 70% ethanol for lh and are carefully shaken to get clear before the cell culture. The dried scaffold is then immersed in the culture medium (its composition is described below). After 24h, the medium is exchanged for a fresh one.

The cells are isolated from small pieces of the donor's bone tissue (explants) through mechanical purification, in order to remove all tissues except bone, and are cut into pieces ab. the size of 1mm. The bone pieces are then digested in collagenase of 37°C overnight. The tissues which were digested are then removed, whereas the bone pieces are washed in PBS, and then located in culture bottlers in a culture medium of the following composition: DMEM (GIBCO BRL catalog no.22320), enriched with inactivated fetal calf serum of a 10% concentration, with the addition of an antibiotic in the form of Antibiotic- Antimycotic (GIBCO, catalog no. 15240096, consisting of 10.000 U of penicillin in the form of natrium salt of penicillin G, 10.000μg of streptomycin in the form of streptomycin sulphate), and 25μg amphotericin B/ml - in the form of Fungizone® of 0.85% in saline) at the concentration of 1%, L-glutarnine at the concentration of 2mM (GIBCO BRL) and vitamin C at the concentration of 120 μΜ in the form of L-ascorbic acid 2-phosphate (SIGMA)). The procedure is performed in sterile conditions. The culture is performed in an incubator of a constant humidity above 95%, temperature of 37°C, and the presence of 5% carbon dioxide. After reaching confluence, the cells are deatched from the surface by trypsin digestion; then, the cells are centrifuged, counted and resuspended in the mentioned culture medium.

The scaffolds are placed in sterile culture dishes. The cells suspended in the culture medium are placed dropwise on the scaffold at the concentration of 50000 cells/scaffold. The cell culture lasts for 48h, and then the medium is exchanged for medium described above, enriched with dexamethasone (lOnM ml) and β-phosphoglicerol (lOmM). Biochemical measurements of the lactic acid concentration released by the scaffold, are performed after 3, 7, 14 and 21 days of culture (Fig. 9). The Number of osteoblastic cells is controlled at the same time points (Fig. 10). The amount of osteocalcin, the marker of mature osteoblastic cells, is measured in the 2. and 3. week of culture (Fig. 11).

The progressive growth of tissue is measured by an assesment of the collagen amount on the scaffolds after 7, 14 and 21 days of culture (Fig. 12). The measurements involves a determination of the hydroxiporline content in the hydrolizates of the products.

The cells and the extracellular matrix fill the scapces between the scaffold fibers (Fig. 13). Example 6. The activity of lactic acid added to the culture medium on osteogenic cells isolated from human bone.

The cells are isolated from small pieces of the donor's bone tissue (explants) through mechanical purification, in order to remove all tissues except bone, and cut into pieces ab. the size of 1mm. The bone pieces are then digested in collagenase of 37°C overnight. The tissues which were digested are then removed, whereas the bone pieces are washed in PBS and then located in culture bottlers in a culture medium of the following composition: DMEM (GIBCO BRL catalog no.22320), enriched with inactivated fetal calf serum of 10% concentration, with the addition of an antibiotic in the form of Antibiotic- Antimycotic (GIBCO, catalog no. 15240096, consisting of 10.000 U of penicillin in the form of natrium salt of penicillin G, 10.000μg of streptomycin in the form of streptomycin sulphate) and 25μg amphotericin B/ml - in the form of Fungizone® of 0.85% in saline) at the concentration of 1%, L-glutamine at the concentration of 2mM (GIBCO BRL), and vitamin C at the concentration of 120μΜ in the form of L-ascorbic acid 2-phosphate (SIGMA)). The procedure is performed in sterile conditions. The culture is performed in an incubator of a constant humidity above 95%, temperature of 37°C and the presence of 5% carbon dioxide. After reaching confluence, the cells are detached from the surface by trypsin digestion; then, the cells are centrifuged, counted, and resuspended in the mentioned culture medium.

The cells are placed at the concentration of 30000/cm into the sterile wells of the culture dishes. The culture takes place in conditions described above. After 48h, the medium is exchanged for a medium described above, enriched with dexamethasone (lOnM/ml) and β-phosphoglicerol (lOmM) and L-lactic acid at the concentrations: 0, 6.25, 7.5, 12.5, 15, 25, 30, 50, 60, 100 mM for the given experimental groups. The culture lasts for 21 days; the enriched culture medium is exchanged 2 times a week. Quantitative assays are performed after 3, 7, 14 and 21 days of culture: the viability of cells (XTT assay), the cell number (counted form the DNA amount, PicoGreen assay), enzymatic activity of the alkaline phosphatase, the amount of osteocalcin in the supernatant (ELISA), the amount of collagen type I (ELISA), the amount of mRNA for the selected proteins (collagen type I, alkaline phosphatase, osteoclacin) in comparison to the GAPDH expression.

Lactic acid added to the culture medium at a concentration higher than 30mM is toxic for osteogenic cells, which is demonstrated by the XTT assay and the DNA content. The cell viability reaches the control or is slightly lower that the control when lower concentrations of lactic acid are added. Alkaline phosphatase activity and the expression of its gene is not affected by the lactate presence in the medium. The concentration of osteocalcin and the expression of its gene is higher than in the control when lactate is added. In that case, the amount of collagen type I (measured by ELISA) is higher than in the control (Fig. 14).

The concentration of collagen type I is several times higher at the tested time points after cell stimulation with lactic acid (Fig. 15 - the selected concentration of lactic acid - 25mM). The expression of the collagen type I gene in the osteogenic cells is several times higher at the tested time points after cell stimulation with lactic acid, in comparison to the GAPDH expression (Fig. 16 - the selected concentration of lactic acid - 25mM). Example 7. The activity of lactic acid added to the culture medium on fibroblasts isolated from human connective tissue.

The cells are isolated from small pieces of connective tissue (from the joint cavity) through mechanical purification in order to remove blood vessels and adipose tissue. The obtained tissue is cut into pieces ab. the size of 1mm, and then digested in collagenase of 37°C overnight. Then, the cell suspension is filtered ( (0.2 urn) in order to isolate the fibroblasts. The filtrate is then washed with PBS two times. The cell pellets are located in culture dishes in the culture medium DMEM (GDBCO BRL catalog no.22320), enriched with the inactivated fetal calf serum of a 10% concentration, with the addition of an antibiotic in the form of Antibiotic- Antimycotic (GIBCO, catalog no. 15240096, consisting of 10.000U of penicillin in the form of natrium salt of penicillin G, 10.000μg streptomycin in the form of streptomycin sulphate), and 25 μg of amphotericin B/ml - in the form of Fungizone® of 0.85% in saline), at the concentration of 1%, L-glutamine at the concentration of 2mM (GIBCO BRL). The procedure is performed in sterile conditions. The culture lasts in an incubator of a constant humidity above 95%, temperature of 37°C and the presence of 5% carbon dioxide. After reaching confluence, the cells are detached from the surface by trypsin digestion, then the cells are centrifuged, counted and resuspended in the mentioned culture medium.

The cells are placed at the concentration of 30000/cm into the sterile wells of the culture dishes. The culture takes place in the conditions described above. After 48h, the medium is exchanged for the medium described above, enriched with L-lactic acid at the concentrations of 0 or 25mM for the given experimental groups. The culture lasts for 21 days; the enriched culture medium is exchanged 2 times a week. Quantitative assays are performed after 3, 7, 14 and 21 days: the viability of cells (XTT assay), the cell number (counted form the DNA amount, PicoGreen assay), the amount of collagen type I (ELISA), the total protein content (Pierce assay).

The lactic acid at the concentration of 25mM is not toxic for fibroblasts. The concentration of collagen type I (ELISA) is higher than in the control (Fig. 17). The amount of protein is equal in both cultures.

Example 8. The activity of lactic acid added to the culture medium on fibroblasts isolated from human connective tissue depending on the presence or absence of ascorbic acid in the culture medium.

The cells are isolated from small pieces of connective tissue (from the joint cavity) through mechanical purification, in order to remove blood vessels and adipose tissue. The obtained tissue is cut into pieces ab. the size of 1mm, and then digested in coUagenase of 37°C overnight. Then, the cell suspension is filtered (0.2um), in order to isolate the fibroblasts. The filtrate is then washed with PBS two times. The cell pellets are located in culture dishes in the culture medium DMEM (GIBCO BRL catalog no.22320), enriched with an inactivated fetal calf serum of 10% concentration, with the addition of an antibiotic in the form of Antibiotic- Antimycotic (GIBCO, catalog no. 15240096, consisting of 10.000 U of penicillin in the form of natrium salt of penicillin G, 10.000μg of streptomycin in the form of streptomycin sulphate), and 25μg of amphotericinerycin B/ml - in the form of Fungizone® of 0.85% in saline) at the in concentration of 1%, L-glutamine at the concentration of 2mM (GIBCO BRL). The procedure is performed in sterile conditions. The culture is performed in an incubator of a constant humidity above 95%, temperature of 37°C and the presence of 5% carbon dioxide. After reaching confluence, the cells are detached from the surface by trypsin digestion, then the cells are centrifuged, counted and resuspended in the mentioned culture medium.

The cells are placed at the concentration of 30000/cm into the sterile wells of the culture dishes. The culture lasts in the conditions described above. After 48h, the medium is exchanged for the medium described above, enriched with L-lactic acid at the concentration of 25mM, while ascorbic acid is absent or present in the medium at the concentration of 120μΜ for the given experimental group. The culture lasts for 21 days; the enriched culture medium is changed 2 times a week. The quantitative assay of the DNA content (PicoGreen assay) is performed after 3, 7, 14 and 21 days of culture.

Ascorbic acid added to the culture medium with lactic acid is essential for the maintenance of the cell viability in the prolonged culture. The cell number (measured by PicoGreen assay) at several time points is presented in Fig. 18. Example 9. The use of tissue engineered product to improve the bone conditions prior to the dental implant insertion in the back parts of the maxilla.

The treatment is carried out in local or general anesthesia. A horizontal cut is performed at the top of the alveolar process distal to the last existing tooth. It is permissible to conduct the dismissing vertical cuts forming trapezoidal muco-periosteal flaps on both sides of the alveolar process. After unveiling the bone, the cradle for the planned implants (in accordance with the protocol applicable to the selected implant system) is prepared. The only exception from the standard protocol is the depth of the bone preparation , which should end at about 1-1.5 mm from the sinus floor.

After the cradle is prepared, the trephine is inserted, and, using a hammer, a controlled surgical bone fracture of the sinus floor is made. The preservation of the continuity of the sinus mucosa is essential. Then, using a balloon inserted in the cradle, the mucosa membrane is separated from the bone margin of the sinus floor, using pressure. The created space is then filled with the tissue engineering product using an applicator. The last step is the introduction of the planned implant and suturing of the wound. Loading of the implants is carried out according to the established implantological protocols. It is expected to shorten the time required for the osteointegration of the implants, as a result of the tissue engineering product activity. The determination of the primary and secondary stability of the implants is accomplished using the devices of Periotest and/or Ostell.

In the cases when a significant deficit of bone tissue makes it is impossible to obtain the primary stability of several implants placed at the same time, a modified surgery is acceptable. Only one hole in the central part of the reconstructed alveolar process is prepared. The diameter of the preparation should only allow the introduction of the trephine, and then the balloon applicator. The specific steps in the use of these devices do not change. After the deposit of the tissue engineered product, the hole is protected by a resorbable barrier membrane, and the wound is sutured. After the period of bone remodeling, it is possible to perform the next surgery aimed to insertion the implants. It is expected to shorten the time of the reconstruction of the bone tissue, as a result of the application of the tissue engineered product and an increases the success rate in for the cases of a bone deficit of a significant degree. Example 10 The use of the tissue engineered product in the horizontal bone augmentation after a subperiostal application surgery.

The tissue engineered product is applied using a cannula to the lodge formed between the periosteum and the cortical bone plate. This lodge can be produced using different surgical techniques, preferably by an osmotic expander. After the application of the tissue engineered product, the wound is protected by a resorbable barrier membrane and sutured. After a period of bone remodeling, an increase in the volume of the operated bone is expected.

Example 11 The use of the tissue engineered product for filling soft tissue defects in the procedures of aesthetic medicine.

The tissue engineered product is applied using a needle or cannula percutaneously to fill facial wrinkles and losses, caused by a trauma or other pathologies such as aging of the face and other anatomical regions. The procedure is carried out according to the generally accepted treatment algorithm for the use of fillers in aesthetic medicine. In this case, the filler is a tissue engineered product, and the expected outcomes are enhanced by the introduction of the grafting cells capable of producing extracellular matrix.

Potential clinical application of the product:

The product is to be used clinically to reconstruct bone defects resulting from tumor resection, fracture nonunions, disorders of bone growth, and to fill bone tissue losses after osteolysis within implants, or to augment bone bases for implant fixation. The product may be used for bone reconstruction in maxillofacial surgery, as well as in otolaryngology. The product is to be used clinically to reconstruct soft tissues, as e.g. a filler for subcutaneous tissue in aesthetic surgery, as a burn wound dressing, as a skin substitute, as an alternative to synthetic materials for hernia reconstruction, as a coiling material for vessel defects, and as a material for ligament or tendon reconstruction.

Literature

I. Irwin, R.B., M. Bernhard, and A. Biddinger, Coralline hydroxyapatite as bone substitute in

orthopedic oncology. Am J Orthop (Belle Mead NJ), 2001. 30(7): p. 544-50.

2. Marino, J.T. and B.H. Ziran, Use of solid and cancellous autologous bone graft for fractures and nonunions. Orthop Clin North Am. 41(1): p. 15-26; table of contents.

3. Menick, F.J., Nasal reconstruction. Plast Reconstr Surg. 125(4): p. 138e-150e.

4. O'Fearraigh, P., Review of methods used in the reconstruction and rehabilitation of the maxillofacial region. J Ir Dent Assoc. 56(1): p. 32-7.

5. Pape, H.C., A. Evans, and P. obbe, Autologous bone graft: properties and techniques. J Orthop Trauma. 24 Suppl 1: p. S36-40.

6. Sajjadian, A., R. Rubinstein, and N. Naghshineh, Current status of grafts and implants in

rhinoplasty: part I. Autologous grafts. Plast Reconstr Surg. 125(2): p. 40e-49e.

7. lijn, R., et al., A meta-analysis of histomorphometric results and graft healing time of various biomaterials compared to autologous bone used as sinus floor augmentation material in humans.

Tissue Eng Part B Rev.

8. Shegarfi, H. and O. Reikeras, Review article: bone transplantation and immune response. J Orthop Surg (Hong Kong), 2009. 17(2): p. 206-11.

9. Banwart, J.C., M.A. Asher, and R.S. Hassanein, Iliac crest bone graft harvest donor site morbidity.

A statistical evaluation. Spine (Phila Pa 1976), 1995. 20(9): p. 1055-60.

10. Heneghan, H.M. and J.P. McCabe, Use of autologous bone graft in anterior cervical

decompression: morbidity & quality of life analysis. BMC Musculoskelet Disord, 2009. 10: p. 158.

I I . John, S., et al., A Retrospective Analysis of Anterior Calcaneal Osteotomy with Allogenic Bone Graft. J Foot Ankle Surg.

12. Brandoff, J.F., J.S. Silber, and A.R. Vaccaro, Contemporary alternatives to synthetic bone grafts for spine surgery. Am J Orthop (Belle Mead NJ), 2008. 37(8): p. 410-4.

13. Langer, R. and J.P. Vacanti, Tissue engineering. Science, 1993. 260(5110): p. 920-6.

14. Shen, H., et al., An injectable scaffold: rhBMP-2-loaded poly(lactide-co-glycolide) hydroxyapatite composite microspheres. Acta Biomater. 6(2): p. 455-65.

15. on, E., et al., Autologous bone marrow stromal cells loaded onto porous hydroxyapatite ceramic accelerate bone repair in critical-size defects of sheep long bones. J Biomed Mater Res, 2000. 49(3): p. 328-37.

16. Yamasaki, T., et al., Bone-marrow-derived mononuclear cells with a porous hydroxyapatite scaffold for the treatment of osteonecrosis of the femoral head: a preliminary study. J Bone Joint Surg Br. 92(3): p. 337-41.

17. Yoshimi, R., et al., Self-assembling peptide nanofiber scaffolds, platelet-rich plasma, and

mesenchymal stem cells for injectable bone regeneration with tissue engineering. J Craniofac Surg, 2009. 20(5): p. 1523-30.

18. Qiu, J., et al., [Clinical study on PRP in improving bone repair]. Zhongguo Xiu Fu Chong Jian Wai Ke Za Zhi, 2009. 23(7): p. 784-7.

19. Vacanti, C.A., et al., Replacement of an Avulsed Phalanx with Tissue-Engineered Bone. N Engl J Med, 2001.344(20): p. 1511-1514.

20. Warnke, P.H., et al., Man as living bioreactor: fate of an exogenously prepared customized tissue- engineered mandible. Biomaterials, 2006. 27(17): p. 3163-7.

21. Wozniak, P., et al., Candidate bone-tissue-engineered product based on human-bone-derived cells and polyurethane scaffold. Acta Biomater, 2009.

22. Papadimitropoulos, A.B., A., Wendt, D., Barbero, A., Martin, I., Expansion of human bone marrow stromal cells within porous scaffolds under perfusion. European Cells and Materials, 2007. 14: p. 83.

23. Mizuno, H., Adipose-derived stem cells for tissue repair and regeneration: ten years of research and a literature review. J Nippon Med Sch, 2009. 76(2): p. 56-66.

24. Hou, T., et al., Umbilical cord Wharton's Jelly: a new potential cell source of mesenchymal stromal cells for bone tissue engineering. Tissue Eng Part A, 2009. 15(9): p. 2325-34.

25. In 't Anker, P.S., et al., Isolation of mesenchymal stem cells of fetal or maternal origin from human placenta. Stem Cells, 2004. 22(7): p. 1338-45.

26. Ge, Z., Z. Jin, and T. Cao, Manufacture of degradable polymeric scaffolds for bone regeneration.

Biomed Mater, 2008. 3(2): p. 022001.

27. Athanasiou, K.A., et al., Orthopaedic applications for PLA-PGA biodegradable polymers.

Arthroscopy, 1998. 14(7): p. 726-37.

28. Yu, N.Y., et al., Biodegradable poly(alpha-hydroxy acid) polymer scaffolds for bone tissue

engineering. J Biomed Mater Res B Appl Biomater. 93(1): p. 285-95. 29. Tsuji, H., Poly(lactide) stereocomplexes: formation, structure, properties, degradation, and applications. Macromol Biosci, 2005. 5(7): p. 569-97.

30. Lu, J.M., et al., Current advances in research and clinical applications of PLGA-based

nanotechnology. Expert Rev Mol Diagn, 2009. 9(4): p. 325-41.

31. Barrett, D.G. and M.N. Yousaf, Design and applications of biodegradable polyester tissue scaffolds based on endogenous monomers found in human metabolism. Molecules, 2009. 14(10): p.4022-50.

32. Noga, D.E., et al., Synthesis and modification of functional poly(lactide) copolymers: toward

biofunctional materials. Biomacromolecules, 2008. 9(7): p.2056-62.

33. Li, H., Chang, Jiang, pH-compensation effect of bioactive inorganic fillers on the degradation of PLGA. Composites Science and Technology, 2005. 65(14): p. 2226-2232.

34. Rezwan, K., Chen, Q.Z., Blaker, J.J., Boccaccini, AR., Biodegradable and bioactive porous

polymer/inorganic composite scaffolds for bone tissue engineering. Biomaterials, 2006. 27(18): p. 3413-3431.

35. Ara, M., Watanabe, M., Imai, Y., Effect of blending calcium compounds on hydrolytic degradation of poly(DL-lactic acid-co-glycolic acid). Biomaterials, 2002. 23(12): p. 2479-83.

36. Carpizo, K.H., et al., Pretreatment of poly(l-lactide-co-glycolide) scaffolds with sodium hydroxide enhances osteoblastic differentiation and slows proliferation of mouse preosteoblast cells. Plast Reconstr Surg, 2008. 121(2): p. 424-34.

37. Green, D., Walsh, D., Yang, X., Mann, S., Oreffo, R., Stimulation of human bone marrow stromal cells using growth factor encapsulated calcium carbonate porous microspheres. Journal of

Materials Chemistry, 2004.

38. Rhee, S.H., J.Y. Choi, and H.M. Kim, Preparation of a bioactive and degradable polyfepsilon - caprolactone)/silica hybrid through a sol-gel method Biomaterials, 2002. 23(24): p. 4915-21.

39. Green, D. W., et al., Augmentation of skeletal tissue formation in impaction bone grafting using vaterite microsphere biocomposites. Biomaterials, 2009. 30(10): p. 1918-27.

40. Shen, H., et al., Combining oxygen plasma treatment with anchorage of cationized gelatin for

enhancing cell affinity of poly (lactide-co-glycolide). Biomaterials, 2007. 28(29): p. 4219-30.

41. Zhao, J.H., et al., Improving the cell affinity of a poly(D,L-lactide) film modified by grafting

collagen via a plasma technique. Biomed Mater, 2006. 1(4): p. 247-52.

42. Janorkar, A.V., et al., Grafting amine-terminated branched architectures from poly(L-lactide) film surfaces for improved cell attachment. J Biomed Mater Res B Appl Biomater, 2007. 81(1): p. 142- 52.

43. Woo, K.M., V.J. Chen, and P.X. Ma, Nano-fibrous scaffolding architecture selectively enhances protein adsorption contributing to cell attachment. J Biomed Mater Res A, 2003. 67(2): p. 531-7. 44. Lahiri, D., et al., Carbon nanotube reinforced polylactide-caprolactone copolymer: mechanical strengthening and interaction with human osteoblasts in vitro. ACS Appl Mater Interfaces, 2009. 1(11): p. 2470-6.

45. Woo, K.M., et al., Nano-fibrous scaffolding promotes osteoblast differentiation and

biomineralization. Biomaterials, 2007. 28(2): p. 335-43.

46. Badami, A.S., et al., Effect of fiber diameter on spreading, proliferation, and differentiation of osteoblastic cells on electrospun poly(lactic acid) substrates. Biomaterials, 2006. 27(4): p. 596-606.

47. Ifkovits, J.L., H.G. Sundararaghavan, and J.A. Burdick, Electrospinning fibrous polymer scaffolds for tissue engineering and cell culture. J Vis Exp, 2009(32).

48. Jang, J.H., O. Castano, and H. W. Kim, Electrospun materials as potential platforms for bone tissue engineering. Adv Drug Deliv Rev, 2009. 61(12): p. 1065-83.

49. Holy, C.E., et al., Optimizing the sterilization of PLGA scaffolds for use in tissue engineering.

Biomaterials, 2001. 22(1): p. 25-31.

50. Montanari, L., et al., Gamma irradiation effects on poly(DL-lactictide-co-glycolide) microspheres. J Control Release, 1998. 56(1-3): p. 219-29.

51. Cordewene, F.W., et al., Cytotoxicity of poly(96L/4D-lactide): the influence of degradation and sterilization. Biomaterials, 2000. 21(23): p. 2433-42.

52. Cotton, N.J., Egan, M. J., Brunelle, J. E., Composites of poly(DL-lactide-co-glycolide) and calcium carbonate: in vitro evaluation for use in orthopedic applications. J Biomed Mater Res A, 2008. 85(1): p. 195-205.

53. Lam, K., Nieuwenhuis, P., Molenaar, I., Esselbrugge, H., Feijen, J., Dijkstra, PJ., Schakenraad, JM..

Biodegradation of porous versus non-porous poly(L-lactic acid) films. J Mater Sci Mater Med, 1994. 5: p. 181-189.

54. Dong, Y., et al., Degradation behaviors of electrospun resorbable polyester nanofibers. Tissue Eng Part B Rev, 2009. 15(3): p. 333-51.

55. Oh, S.H., S.G. Kang, and J.H. Lee, Degradation behavior of hydrophilized PLGA scaffolds

prepared by melt-molding particulate-leaching method: comparison with control hydrophobic one. J Mater Sci Mater Med, 2006. 17(2): p. 131-7. 56. Shive, M.S. and J.M. Anderson, Biodegradation and biocompatibility of PLA and PLGA microspheres. Adv Drug Deliv Rev, 1997. 28(1): p. 5-24.

57. Bostman, O., Hirvensalo, E., Makinen, J., Rokkanen, P., Foreign-body reactions to fracture fixation implants of biodegradable synthetic polymers. J Bone Joint Surg Br, 1990. 72(4): p. 592-6.

58. Martin, C, Winet, H., Bao, J. Y., Acidity near eroding polylactide-polyglycolide in vitro and in vivo in rabbit tibial bone chambers. Biomaterials, 1996. 17(24): p. 2373-2380.

59. Mikos, A.G., et al., Host response to tissue engineered devices. Adv Drug Deliv Rev, 1998. 33(1-2): p. 111-139.

60. Hutmacher, D.W., Scaffolds in tissue engineering bone and cartilage. Biomaterials, 2000. 21(24): p.

2529-43.

61. Dong, Y., et al., Distinctive degradation behaviors of electrospun polyglycolide, poly(DL-lactide- co-glycolide), and poly(L-lactide-co-epsilon-caprolactone) nanofibers cultured with/without porcine smooth muscle cells. Tissue Eng Part A. 16(1): p. 283-98.

62. Zund, G., et al., The in vitro construction of a tissue engineered bioprosthetic heart valve. Eur J Cardiothorac Surg, 1997. 11(3): p. 493-7.

63. Ma, P.X. and R. Langer, Morphology and mechanical function of long-term in vitro engineered cartilage. J Biomed Mater Res, 1999. 44(2): p. 217-21.

64. Seidel, J.O., et al., Long-term culture of tissue engineered cartilage in a perfused chamber with mechanical stimulation. Biorheology, 2004. 41(3-4): p. 445-58.

65. eogh, M.B., O.B. FJ, and J.S. Daly, A novel collagen scaffold supports human osteogenesis— applications for bone tissue engineering. Cell Tissue Res. 340(1): p. 169-77.

66. Neuss, S., et al., Long-term survival and bipotent terminal differentiation of human mesenchymal stem cells (hMSC) in combination with a commercially available three-dimensional collagen scaffold Cell Transplant, 2008. 17(8): p. 977-86.

67. Blanck, T.J. and B. Peterkofsky, The stimulation of collagen secretion by ascorbate as a result of increased proline hydroxylation in chick embryo fibroblasts. Arch Biochem Biophys, 1975. 171(1): p. 259-67.

68. Yamamoto, I., et al., Collagen synthesis in human skin fibroblasts is stimulated by a stable form of ascorbate, 2-O-alpha-D-glucopyranosyl-L-ascorbic acid. J utr, 1992. 122(4): p. 871-7.

69. Cai, D.Z., et al., Biodegradable chitosan scaffolds containing microspheres as carriers for

controlled transforming growth factor-betal delivery for cartilage tissue engineering. Chin Med J (Engl), 2007. 120(3): p. 197-203.

70. Gebken, J., et al., Hypergravity Stimulates Collagen Synthesis in Human Osteoblast-Like Cells:

Evidence for the Involvement ofp44/42 MAP-Kinases (ERK 1/2). J Biochem, 1999. 126(4): p. 676- 682.

71. Gladden, L.B., Lactate metabolism: a new paradigm for the third millennium. J Physiol, 2004.

5580* 1): P- 5-30.

72. Ghani, Q.P., et al., Regulatory role of lactate in wound repair. Methods Enzymol, 2004. 381: p.

565-75.

73. Yalamanchi, N., et al., Flexor tendon wound healing in vitro: lactate up-regulation ofTGF-beta expression and functional activity. Plast Reconstr Surg, 2004. 113(2): p. 625-32.

74. Comstock, J.P. and S. Udenfriend, Effect of lactate on collagen proline hydroxylase activity in cultured L-929 fibroblasts. Proc Natl Acad Sci U S A, 1970. 66(2): P- 552-7.

75. Green, H. and B. Goldberg, Collagen and Cell Protein Synthesis by an Established Mammalian Fibroblast Line. Nature, 1964. 204: p. 347-9.

76. Klein, M.B., et al., Flexor tendon wound healing in vitro: the effect of lactate on tendon cell

proliferation and collagen production. J Hand Surg Am, 2001. 26(5): p. 847-54.

77. Spector, J.A., et al., Osteoblast expression of vascular endothelial growth factor is modulated by the extracellular microenvironment. Am J Physiol Cell Physiol, 2001. 280(1): p. C72-80.

78. Hunt, T.K., et al., Aerobically derived lactate stimulates revascularization and tissue repair via redox mechanisms. Antioxid Redox Signal, 2007. 9(8): p. 1115-24.

79. Constant, J.S., et al., Lactate elicits vascular endothelial growth factor from macrophages: a

possible alternative to hypoxia. Wound Repair Regen, 2000. 8(5): p. 353-60.

Claims

Patent claims

1. The method for obtaining a tissue engineered product for the reconstruction and regeneration of bone tissue, prepared by cmturing viable cells of an osteogenic potential or other cells producing collagen type I on a biodegradable scaffold of a complex structure, characterized in that the product is ready for implantation after reaching an advanced scaffold degradation, accompanied by an advanced production of the extracellular matrix proteins in vitro, by the cells seeded on the scaffold, prepared from a mixture of polymers/copolymers, or polymers/copolymers modified with fillers, while cells are isolated from the donor's tissue and then cultured in a proper medium for each cell type in vitro, until they reach an appropriate number, preferably 70-80% of confluence in culture; then, the cells are detached from the culture surface, resuspended in the proper medium, and counted, whereas the scaffolds immersed in the culture medium are located in a device with a constant flow of the culture medium, or they are located in the culture dishes; then, the suspension of cells in proper concentration is put into the device or into the wells of the culture dish; then, they are located in an incubator of a constant humidity above 95%, temperature of 37°C and the presence of 5% carbon dioxide; then, the culture lasts for a minimum of 7 days, while the culture medium of the same composition is exchanged at least once; then, the products are replaced into the culture dish and cultured further until an advanced degradation of the polymeric material is reached, i.e., when more than 50% of the lactic acid is released from the material - after that, the product is ready for use.

2. The method according to claim 1, characterized in that the substrate is a material releasing lactic acid, particularly polymers and copolymers of the lactic acid, preferably selected from the PLA, D-PLA, L-PLA, LD-PLA, and block copolymers, grafted or statistical, of the above varieties of the PLA with each other and with the PGA, PEG (poly (ethylene glycol)) and PCL, while the lactic acid is released by the substrate in the environment of the culture medium and stimulates osteogenic cells, and other cells capable of the secretion of type I collagen, embedded in the substrate, for the production of collagen type I, which is an essential component of the extracellular matrix.

3. The method according to claim 2, characterized in that as a substrate for the cells are used materials releasing lactic acid and/or modified materials, preferably different varieties of the PLA and its copolymers, preferably selected from the PLA, D-PLA, L-PLA, LD-PLA, and block copolymers, grafted or statistical, of the above varieties of the PLA with each other and with the PGA, PEG, and PCL, with fillers preferably in the form of dispersed powder of crystalline or amorphous salts of lactic acid, preferably selected from calcium lactate, sodium lactate, potassium lactate, and magnesium lactate, being an additional source of lactic acid.

4. The method according to claim 2, characterized in that as a substrate for the cells are used materials releasing lactic acid and/or modified materials, preferably different varieties of PLA and its copolymers, preferably selected from the PLA, D-PLA, L- PLA, LD-PLA, and block copolymers, grafted or statistical, of the above varieties of the PLA with each other and with the PGA, PEG (poly (ethylene glycol)), and PCL, preferably with ceramic fillers in the form of nanoparticles or microparticles in the form of powder or ceramic granules, particularly stoichiometric and nonstoichiometric calcium phosphates, calcium carbonates, silicon oxides, and multiphase materials of these groups.

5. The method according to claim 2, characterized in that as a substrate for the cells are used materials releasing lactic acid and/or materials with modifications in the form of additives releasing growth factors, while the polymers and copolymers are preferably with a filler in the form of powder or granules of a demineralized bone matrix.

6. The method according to claim 2, characterized in that the embedded cells are cells of an osteoblastic potential, in particular cells isolated from bone tissue or progenitor cells, including those isolated from adipose tissue, bone marrow, peripheral blood, foetal tissues or cells of an angiogenic potential, in particular cells isolated from the endothelium, progenitor cells or cells of an osteoclastic phenotype.

7. The method according to claim 6, characterized in that the cells embedded in one scaffold may be cells of a different phenotype and a different potential.

8. The method according to claim 6 or 7, characteristized in that the seeded cells are preferably cells isolated from mammalian tissues, preferably human, demonstrating species compatibility, and, in the case of its application in a human, preferably as autologous graft.

9. The method according to claim 1, characterized in that the cells building the graft are any cells able to synthesize the extracellular matrix characteristic for connective

^• tissue. ' .

10. The method according to claim 2, characterized in that collagen fibers are formed, which are the base for the proliferation of cells, whose activity intensifies the scaffold degradation, causing a further release of lactic acid and, due to the presence of the lactic acid, a further stimulation of collagen type ί and extracellular matrix contribution in the product, which causes a substitution of the original scaffold with tissue obtained in in vitro conditions before introducing into the recipient's body.

11. The method according to claim 2, characterized in that the lactic acid released by the lactate-based scaffold stimulates and controls the rate of collagen fiber formation, and the scaffold shape determines the three-dimensional fiber architecture of the collagen produced by the seeded cells in the cell culture.

12. The method according to claim 2, characterized in that the lactic acid concentration does not exceed the toxic concentration for the given cells.

13. The tissue engineered product for the reconstruction and regeneration of bone tissue, obtained using the method according to claims 1 to 12, prepared by culturing viable cells of an osteogenic potential or other cells producing collagen type I on a biodegradable scaffold of a complex structure, characterized in that the product is ready for implantation after reaching an advanced scaffold degradation, accompanied by an advanced production of extracellular matrix proteins in vitro, by cells seeded on the scaffold.

14. The product according to claim 13, characterized in that the substrate is a material releasing lactic acid and/or modified material, preferably particularly polymers and copolymers of the lactic acid, preferably selected from the PLA, D-PLA, L-PLA, LD-PLA, and block copolymers, grafted or statistical, of the above varieties of the PLA with each other and with the PGA, PEG, and PCL, while the lactic acid is released by the substrate in the environment of the culture medium and stimulates the osteogenic cells and other cells capable of the secretion of type I collagen, embedded in the substrate for producing collagen type I, which is the basic component of the extracellular matrix.

15. The product according to claim 14, characterized in that as a substrate for the cells is used a material releasing lactic acid and/or modified material, preferably different varieties of the PLA and its copolymers, preferably selected from the PLA, D-PLA, L-PLA, LD-PLA, and block copolymers, grafted or statistical, of the above varieties of PLA. with each other and with the PGA, PEG and PCL, with fillers preferably in the form of a dispersed powder of crystalline or amorphous salts of the lactic acid, preferably selected from calcium lactate, sodium lactate, potassium lactate, and magnesium lactate, being an additional source of lactic acid.

16. The product according to claim 14, characterized in that the substrate for the cells is a material releasing lactic acid and/or modified material, preferably different varieties of the PLA and its copolymers, preferably selected from the PLA, D-PLA, L-PLA, LD-PLA, and block copolymers, grafted or statistical, of the above varieties of the PLA with each other and with the PGA, PEG (poly (ethylene glycol)) and PCL, preferably containing ceramic fillers in the form of nanoparticles or microparticles in the form of powder or ceramic granules, particularly stoichiometric and nonstoichiometric calcium phosphates, calcium carbonates, silicon oxides, and multiphase materials of these groups.

17. The product according to claim 14, characterized in that the embedded cells are cells of an osteoblastic potential, in particular cells isolated from bone tissue or progenitor cells, including those isolated from adipose tissue, bone marrow, peripheral blood, foetal tissues or cells of an angiogenic potential, in particular cells isolated from the endothelium, progenitor cells or cells of an osteoclastic phenotype.

18. The product according to claim 14, characterized in that the cells embedded in one scaffold may be cells of a different phenotype and a different potential.

19. The product according to claim 14, characterized in that the seeded cells are preferably cells isolated from mammalian tissues, preferably human, demonstrating species compatibility, and, in the case of its application in a human, preferably as autologous graft.

20. The product according to claim 14, characterized in that the cells building the graft are any cells able to synthesize the extracellular matrix characteristic for connective tissue.

21. The product according to claim 14, characterized in that collagen fibers are formed, which are the base for the proliferation of cells, whose activity intensifies the scaffold degradation, causing a further release of lactic acid, and, due to the presence of the lactic acid, a further stimulation of collagen type I and extracellular matrix contribution in the product, which causes a substitution of the original scaffold with tissue obtained in in vitro conditions before the implantation into the recipient's body.

22. The product according to claim 14, characterized in that the lactic acid released by the lactate-based scaffold stimulates and controls the rate of collagen fiber formation, and the scaffold shape determines the three-dimensional fiber architecture of the collagen produced by the seeded cells in cell culture.

23. The product according to claim 14, characterized in that the lactic acid concentration does not exceed the toxic concentration for the given cells.

24. The product according to claim 14, characterized in that the product consists of a ntinimum of 80% natural components produced by the cells seeded on the scaffold.

25. The product according to claim 14, characterized in that the cells come from the donor who is the recipient of the graft, which is composed only of the patient's own biological material so that immunological compatibility is provided.

26. The product according to claim 14, characterized in that the product possesses autologous graft features.

27. The use of the tissue engineered product described in claims 13 and 26, wherein the product is implanted after reaching an advanced scaffold degradation accompanied by an advanced production of extracellular matrix proteins in vitro, to be implanted in bone tissue or soft tissue defects, depending on type of cells used and/or to create specific tissue.

28. The use according to claim 27, wherein the product is applied in the regeneration of bone tissue in maxillary sinus floor elevation.

29. The use according to claim 27, wherein the product is applied to produce bone after subperiosteal application.

30. The use according to claim 27, wherein the product is applied in the regeneration of soft tissue in the procedures in aesthetic medicine.