KR20050023298A - Orthopaedic materials derived from keratin - Google Patents

Abstract

The present invention provides biocompatible materials derived from keratin useful in many aspects of medical treatment of bone. The keratin materials may be porous for use as a bone replacement and augmentation product, but also the use of dense keratin materials in bone therapy for use as an internal fixation device in the treatment of fractures and bone regeneration, as well as preserving and restoring bone form and function And methods of making keratin materials for use in development.

Description

Orthopedic materials derived from keratin {ORTHOPAEDIC MATERIALS DERIVED FROM KERATIN}

The present invention relates to the manufacture of medical materials from keratin derived from sources of animals such as wool, hair, horns, hoofs, and scales. The keratin materials described are biocompatible, biointegratable and biodegradable, and the main application of the materials is in orthopedic surgery for replacement and augmentation of bone, and fixation and immobilization of fractures and bone fragments.

Keratin is a class of structural proteins that are present extensively in biological structures, particularly epithelial tissue of higher vertebrates. Keratin can be subdivided into two main classes: soft keratin (occurring in the skin and some other tissues) and hard keratin (forming materials of nails, toenails, hair, horns, feathers and scales).

The toughness and insolubility of hard keratin, allowing it to play an essential structural role in many biological systems, is a desirable feature found in many industrial and consumer products derived from synthetic polymers. In addition to retaining good physical properties, keratin as a protein is a polymer with a high degree of chemical functionality and consequently exhibits a number of properties that synthetic polymers cannot achieve. Thus, keratin is well suited for the development of medical products with high value niche applications. Medical materials that are absorbed (reabsorbed) by body tissues after functioning are examples of high-value product fields in which certain characteristics of keratin allow them to outperform both natural and synthetic competition materials.

Yamauchi (K. Yamauchi, M. Maniwa and T. Mori, Journal of Biomaterial Science, Polymer edition, 3, 259, 1998) can be processed into a matrix in which keratin is considered biocompatible by in vitro and in vivo properties. It is arguing. The processing methods used to make these materials require high concentrations of reducing agents such as thiols, and processing conditions that are not suitable for commercial material production.

Kelly (WO 03/19673) shows that keratin can be processed into complex shapes using commercially viable chemistry and processing conditions.

Blanchard (US 5,358,935 and US 2003 / 0035820A1) demonstrate that keratin can be extracted and processed from human hair using high concentrations of reducing agents, or stringent oxidizing agents, to produce materials useful for some soft tissue applications. However, extraction and reconstitution methods are rigorous and lead to the breakdown of keratin by irreversible oxidation of the unique keratin amino acid cystine to cysteinic acid or by exposure of the protein to high pH conditions that will result in peptide hydrolysis. This results in the loss of many of the beneficial properties of the protein, particularly the toughness necessary for hard tissue application.

In order to produce keratin biomaterials suitable for orthopedic applications, there is a need for processing methods that maintain keratin characteristics and provide the material with good toughness properties. The present invention describes such materials and production methods.

US 6,432,435 claims a tissue engineering backbone with keratin with a hydrophilic group, where the keratin is bound by keratin-keratin disulfide bonds. However, the patent does not disclose how sulfonated keratin can be incorporated into hard tissues such as bone. The examples provided therein are all directed to use in soft tissues or porous structures.

The present invention has found that keratin can be incorporated into hard tissues such as bone and thus used in the treatment of bone damage.

Many tissues in the body, including bones, are constantly being regenerated. The new bone matrix (mineralized to be mineralized) is laid down by certain cells, mainly called osteoblasts, and different components of the bone are removed by osteoclasts. Implants that are removed by these biological processes and replaced with bone tissue have greater advantages over other mechanisms in the body, such as those degraded by chemical degradation. The new bone is formed in parallel with the surface of the implant material, thereby integrating it into the tissue until it is completely reabsorbed and replaced.

Bone can be divided into four microstructural components: cells, organic matrix, inorganic matrix, and soluble signaling factors. Osteoclasts are metabolically active secretory cells expressing soluble signaling factors and osteoids, products that yield an organic insoluble underlayer consisting mostly of type I collagen by extracellular modification. Expression of these products by osteoblasts occurs during maintenance (eg, remodeling), and bone repair. The monocyte-macrophage precursor found in the bone marrow enters the circulatory state and, via asynchronous fusion, produces multinuclear cells up to 100 microns in diameter with an average of 10 to 12 nuclei, known as osteoclasts. Osteoclasts have corrugated boundaries and these points constitute the resorption zone of osteoclasts where enzymatic degradation of the bone surface occurs. The term "remodeling" is used to describe dynamic events associated with bone marrow repair and homeostasis in adults. The summation of the processes associated with homeostasis remodeling is known as activation-resorption-forming. When osteoblasts are activated by signaling factors leaving the area of bone; Osteoclasts begin to stimulate, and osteoblasts enter the empty areas where they leave, attach, reabsorb, and stop reabsorption and abandon attachment in response to certain signals that have not yet been identified. Osteoclast resorption pit begins to repopulate by osteoblastic detachments expressing the ashes that calcify the bone being repaired. In humans, the activation-resorption-forming process occurs between three and six months.

There is a massive bleeding behind injury to the bone (eg, surgical removal of a fracture or tumor), and within 2 to 5 days the bleeding forms a massive blood clot. Neovascularization begins to develop around these blood clots. In addition, a standard inflammatory response occurs in the surrounding soft tissue resulting in the accumulation of polymorphonuclear leukocytes, macrophages, and monocyte cells around the blood clot. By the end of the first week, most blood clots are organized by blood vessel invasion and early fibrosis. The first bone (woven bone) is formed after 7 days. Since bone formation requires a smooth blood supply, the occlusal bones begin to form around the blood clots where vascular formation is greatest. Pluripotent mesenchymal cells from the surrounding soft tissue and from inside the bone marrow give rise to osteoblasts that synthesize the occlusal bone. Frequently cartilage is also formed and ultimately replaced by intrachondral ossification. Granulation tissue containing bone-cartilage is called callus (salt).

After the first week, subsequent steps are initiated and extended for several months depending on the degree of exercise and fixation. By this stage, acute inflammatory cells are dissipated and a repair process commences with the differentiation of pluripotent cells into fibroblasts. Repair proceeds from the periphery to the center and accomplishes two purposes: one is to organize and reabsorb blood clots; The second is more important, providing neovascularization for the construction of the bone, ultimately bridging the bone-deficient site. The events that lead to the repair are: Many osteoclasts from the surrounding bone migrate to the site where healing occurs. The renal vessels are accompanied by these cells to nourish and provide more pluripotent cells for cell regeneration. The site is remodeled by osteoclasts (recovery phase).

After a few weeks, the osteotomy sutures the bone end and remodeling begins, where the bone is reorganized and the original cortex is restored (remodeler).

Purpose of the Invention

It is an object of the present invention to provide materials that contain keratin and are useful for assisting bone formation and / or provide a useful choice for the public.

Summary of the Invention

The present invention provides keratin-containing materials for use in the preservation, repair and development of bone morphology and function in the skeletal system.

The present invention also provides a porous keratin material for use in replacing and augmenting bone.

The present invention also provides a dense keratin material for use in bone fixation and passivation.

Keratin is preferably S-sulfonated and more preferably enriched in intermediate filament proteins

Dense keratin materials may be prepared by compacting solid keratin powders or by compacting keratin films.

The material may contain up to 60% calcium salt.

The material may be prepared by compression and lyophilization of solid keratin.

Alternatively, the solution of keratin may be lyophilized.

The invention also provides the use of keratin materials in the preservation, repair and development of bone morphology and function.

The present invention also provides

a) compacting keratin in the presence of soluble porogen;

b) removing the porogen and strengthening the material;

c) washing the protein material; And

d) freeze drying the material;

Provided are methods of forming porous S-sulfonated keratin enriched materials.

The present invention also provides a method of forming a keratin material into an orthopedic product.

The invention also provides a biocompatible material in the form of porous keratin that is concentrated in intermediate filament proteins for use in bone replacement / enhancement therapy.

The material may be prepared by the compression of the solid keratin powder in the presence of porogen, followed by freeze drying after the compression. Porogens may be selected from sodium chloride or another biocompatible salt, or glycerol or another biocompatible solvent. The amount and type of porogen can be controlled to select the pore size and to allow the infiltration of bone progenitor cells at the time of transplantation to promote the colonization of keratin materials.

The invention also

a) compressing keratin in the presence of heat and water;

b) strengthening the material;

c) washing the material to remove residual chemicals; And

d) drying the material,

A method of forming a dense material of S-sulfonated keratin materials into orthopedic products.

The invention also

a) strengthening the keratin-containing starting material;

b) washing the material to remove residual chemicals;

c) drying the material; And

d) compacting the keratin in the presence of heat and water,

A method of forming a dense material of S-sulfonated keratin into an orthopedic product.

The present invention also provides orthopedic medical materials such as plates, pins and screws made from biocompatible keratin materials for the treatment of fractures by internal fixation and fixation and immobilization of bone fragments.

The present invention also provides a method for reforming S-sulfonated keratin concentrated in intermediate filament proteins into a tough and dense biocompatible material for use as an internal fixation device in the treatment of fractures.

Water and chemicals may be added. Controlled use of a reducing agent can be used to remove sulfonate groups from S-sulfonate keratin and to reconstitute the disulfide originally present.

The keratin used for bone formation can be prepared by the method according to WO 03/011894, which provides a process for the preparation of high molecular weight keratin derivatives, according to which the method is used to remove the keratin source by oxidative sulfitolysis. A first stage digestion step of sulfonation, a second stage repeat aqueous extraction with separation of soluble and insoluble keratin, and subsequently re-extracting insoluble keratin to produce a highly S-sulfonated keratin derivative.

WO 03/018673 provides foam fiber adhesive materials or films derived from S-sulfonated keratin proteins. The method involves solvent casting a solution of S-sulfonated keratin protein.

Also provided are methods of making films, foams or adhesive materials derived from highly S-sulfonated keratin intermediate filament proteins.

The present invention provides a method of making keratin materials suitable for use in bone structures and also provides keratin materials for use in bone structures based on the methods of WO 03/018673 and WO 03/011894.

Degradable keratin devices will provide sufficient flexibility for promoting bone growth (unlike some hard permanent materials currently in use) by progressively delivering functional loads to the healing bone. In addition, no subsequent surgery is required to remove the keratin-derived fixation device or device.

Reconstruction of S-sulfonated keratin, concentrated in intermediate filament proteins, into a tough, dense material for use as an internal fixation device in fracture therapy involves the compression of biocompatible proteins in the presence of moisture, chemicals, and in some cases heat. It may include the process of forming the desired shape. The formation of crosslinks in the material to ensure strength and toughness under biological conditions occurs during compaction or subsequently through chemical treatment.

The method may also involve removing sulfonate groups from S-sulfonated keratin and reforming the disulfide originally present in native keratin through controlled use of reducing agents. This process firstly provides strength and toughness under biological conditions by effectively polymerizing keratin proteins through an extended network of disulfide crosslinks; Second, it serves a dual purpose of regulating the rate and extent of biodegradation that occurs by disrupting the enzymatic digestion of materials. By controlling the rate of biodegradation, the present invention allows keratin products to be used in applications where a range of healing periods are desired.

The reconstituted keratin described in this patent specification is completely biocompatible and does not elicit any significant foreign body type immune response. Keratin is broken down and replaced with normal bone by the normal processes occurring in the bone described above. Thus, in bone replacement and augmentation, keratin has properties similar to autologous bone. In the case of fracture treatment, such keratin-made instruments will possess the necessary physical properties to perform fixation and immobilization of bone fragments, and once this function is completed, the keratin will be gradually reabsorbed and ultimately disappeared from the tissue. do. During the initial period of resorption, the progressively weakening keratin machinery promotes bone formation by putting bone into increasing functional load. This process will prevent the stress shielding effect found with metal instruments. In addition, the degradation and resorption of keratin is a major advantage over metal instruments that are permanently present in the body or require secondary surgical procedures for their removal.

The present invention can be applied to any bone forming function. The shape and function of the bone can be disrupted by:

Developmental abnormalities in which the bones are underdeveloped or abnormally developed,

Trauma causing fracture or fracture dislocation, without bone loss,

Surgical interventions such as incisions in malignant or benign tumor / tumor-like symptoms, or degenerative symptoms of bone,

-Bone necrosis and destruction caused by symptoms such as infection

Therapeutic modalities for these symptoms seek to preserve, repair, and develop the normal form and function of diseased bone in the skeletal system.

The term "middle filament protein" describes keratin proteins from the middle filament protein class. As discussed in Marshall et al., Structure and Biochemistry of Mammalian Hard Keratin, Electron Microscopy Review, Vol 4, pp 47-83, 1991, keratin intermediate filaments are derived from hard alpha keratin and are generally Is recognized in the art as including two families of low sulfur polypeptides containing both helical and non-helical fragments.

"Concentrated in intermediate filament protein" means that there are more intermediate filament proteins present in the product than in the corresponding amounts of native keratin sources.

Description of Preferred Embodiments of the Invention

The invention is described below by way of example only with reference to the following preferred embodiments.

Features of the present invention specifically illustrate some methods and applications based on hard α-keratin from wool. However, such principles can equally well apply to any source capable of producing alternative α-keratin, or intermediate filament (IF) type proteins.

Similar preparation methods have been applied by the applicant to other keratin sources, such as feathers, to produce materials that are equally well suited for some of the applications described below. Features of the present invention extend to encompass a limited area of use of such keratin in applications that do not depend on the presence of a type of protein (IF protein). This includes applications where agents based on β-keratin or feather keratin can be incorporated with IF proteins.

Wool represents a convenient source of hard α-keratin, but other animal fibers, or horns, or hoofs, will function equally well as a source of protein of interest. Wool consists of approximately 95% keratin, which can be roughly divided into three protein classes. Intermediate filament proteins are typically high molecular weight (45-60 kD), partially fibrillated tertiary structure and having a cysteine content of about 6%. They make up about 58% of wool fibers by mass, but only a fraction of this mass actually spirals in structure. High and ultra high sulfur proteins, which comprise approximately 26% of the wool fibers, are spherical in structure, have a molecular weight of 10 to 40 kD and can contain cysteine levels up to 30 mole%. High-glycine-tyrosine proteins are a small class of 6% of wool fibers, have molecular weights on the order of 10 kD and are characterized by high content of glycine and tyrosine amino acid residues.

Proteins from different classes of wool keratin possess characteristics that will confer them unique advantages in specific applications.

The present invention generally relates to the use of intermediate filament proteins and to the production of materials for use in orthopedic surgery.

Nevertheless, other non-fibrillary proteins are naturally applicable in more limited applications.

Highly S-sulfonated keratin derived from an animal source as described above is an ideal biocompatible / biodegradable material for use in a range of products for orthopedic applications when purified to separate intermediate filament protein components. . Keratin can be prepared by a method as outlined in WO 03/011894. S-sulfonated keratin proteins themselves can be prepared according to the methods described in WO 03/018673. According to these methods the cystine present in the original keratin source is modified to be S-sulfocysteine. This group is highly polar and can make derivatized keratin soluble under aqueous conditions of pH > In addition, S-sulfonated materials are moisture sensitive and suitable for processing into matrices useful in orthopedic procedures. In some cases it is useful to have S-sulfonate functional groups, while in other matrices, the protein is chemically treated as part of the reconstitution process to remove the S-sulfonate functional groups and remove the disulfide bonds inherent in keratin. It is useful to recover. Since it is highly polar and therefore moisture sensitive, the presence of S-sulfo derivatization makes the reconstituted keratin material susceptible to enzymatic degradation by proteolytic enzymes present in vivo. The conversion of the S-sulfo group to cystine renders the prepared keratin materials insoluble and also imparts some resistance to proteolytic enzymes. Cysteine-containing materials therefore degrade at a slower rate in the body, which is an important performance feature in some orthopedic applications.

The conversion of S-sulfocysteine to cystine can occur through the application of a reducing agent, typically a thiol containing solution such as ammonium thioglycolate, or via a method as described in WO 03/018673.

As a proteinaceous material, keratin is well suited as a material used in the production of orthopedic treatment materials incorporating osteogenic agents such as well-documented osteogenic hydroxyapatite. Depending on the reconstitution method, reconstituted keratin materials containing hydroxyapatite at levels of 0 to 60% can be readily prepared. The beneficial healing effects of including hydroxyapatite in biomaterials are well documented or justified as part of the present invention.

In one aspect of the invention, the S-sulfonated keratin is reconstituted with a porous material for use as a bone replacement or augmentation product. This is accomplished by compacting keratin proteins in the presence of soluble porogens such as sodium chloride and water. In general, S-sulfonated keratin intermediates, preferably 125-200 microns, of S-sulfonated keratin intermediate, ground to a desired particle size to aid powder compaction, are in a ratio of 1: 0.1 to 1:10, preferably 1: 1, And also sodium chloride in a ratio of 1: 0.01 to 1:10. Hydroxyapatite is also incorporated into the mixture at a level of 0 to 60% by mass, preferably 0 to 10% by mass. The mixture is charged to a die and pressurized for 1 to 30 minutes at a pressure in the range of 10,000 to 50,000 kPa. Upon removal from the die, the cylinder is cut from the pellet and the cylinder is immersed in a chemical treatment solution to wash out the porogen and leave a porous material, while also removing sulfonate functionality from keratin and restoring disulfide crosslinking to the protein. Such chemical treatment may take one of two forms. First, the cylinder is treated for 2 to 48 hours, preferably 18 hours, using a solution of ammonium thioglycolate, preferably 0.25 M, containing 0.1 M sodium phosphate buffered to pH 7.0. can do. Residual chemicals are removed by subsequent washing of the pellets in water. After washing, the cylinder is lyophilized. Alternatively, the cylinder may be subjected to chemical treatment for 2 to 48 hours, preferably 18 hours in a solution containing thioglycolic acid, preferably 0.1 M. After a short wash in water, residual chemicals are removed by washing the cylinder in a buffer solution for 48 to 96 hours. Replenish the solution every 24 hours. After further washing in water, the cylinder is lyophilized.

The pore size in the material can be controlled by varying the amount of sodium chloride used in the preparation. For example, typical pore sizes for preparation using 0.06 g sodium chloride per g protein are 50 to 150 microns, while 0.14 g sodium chloride per g protein produces less than 320 microns pores.

The biological properties of porous keratin materials prepared using thioglycolic acid methods are demonstrated both in vitro and in vivo. In vitro, the material is not cytotoxic and supports the growth of human and sheep fibroblasts. Using both fibroblasts, direct contact of the porous material with the cells, as described in ISO 10993-5, produces the following effects. Wells of polystyrene cell culture plates with or without porous material as a control were initially seeded at about 10,000 cells (0 hours). During the first 24 hours after seeding, the cultures had a lag time as evidenced by a decrease in cell number. This phenomenon was recognized in all analyzes performed and a drop was observed in the control wells as well as those containing the test material. Experiments have shown that this lag time lasts less than 12 hours and exponential growth begins at this point. Population doubling occurs semi-adhesive (approximately 80% adhesion) marking the end of the logarithmic growth phase approximately every 24 to 48 hours. This corresponds to the end of the experiment period (5 days or 120 hours). An extended period of experiment indicated a plateau in cell proliferation immediately after this with complete adherence of the culture. Contact inhibition and nutrient depletion play a major role in limiting the rate of proliferation at this point and monolayer cultures show signs of cell death (ie loss of membrane preservation, decrease in cell number, fear formation of individual cells). During the analysis, the cells were observed to attach to the upper surface of the disk. According to light microscopy, the morphological appearance of these cells was considered similar on all substrates compared to the non-material control. Similar analyzes with human fibroblasts showed very similar results with typical fibroblast proliferation curves occurring in the presence of porous materials, reaching approximately 80% adhesion after 120 hours of culture. Control wells reached 100% adhesion at this point.

In vivo studies consisted of keratin materials treated by the method, and also 6% hydroxyapatite, prepared as rods 3 mm in diameter and 3 mm in length, sterilized by gamma irradiation (2.8 Mrad) and adult hind limbs. And a composite of the material that is implanted into the central portion of the long bone (cortical bone) and the proximal and distal ends (cortical-cavernosal bone) of. Tissue responses to the implanted material were studied by histological examination of biopsy samples at days 10, 3, 6, 8, 12 and 24 weeks. Bone tissue for materials made using thioglycolic acid showed a minimal foreign immune response to the presence of the implant and only a thin layer of granulation tissue (fibrosis induction) formed between the implant and the surrounding bone. Within three to six weeks, the implant material is metastasized by the bone tissue to induce the placement of the occlusal bone in the space created by resorption of the keratin implant material, and the renal bone joins into the surrounding bone about six to eight weeks. From week 6, outward remodeling of the occlusal bone into the cortical spongy bone occurred. This process continued to result in full integration of the implant material replaced by mature bone, and bone defects were fully healed.

In addition, the physical properties of these porous materials were prepared in the form of plates having a length of 12 mm, a width of 4 mm, and a depth of 3 mm and irradiated subcutaneously in sheep. Data obtained by testing the plates on an Instron machine showed that the tensile properties were attenuated by approximately 10% over 3-6 weeks. This result is consistent with a dry weight reduction of about 10% at this point. These findings support the new bone formation promotion and stress shielding prevention of the material, and the progressive resorption of the material.

Porous keratin materials are also prepared by freeze drying an aqueous solution of S-sulfonated keratin using a method as described in WO 03/018673, which materials are suitable for use in bone graft applications. Porous keratin materials containing a high proportion of hydroxyapatite preferably contain insoluble hydroxyapatite in an aqueous solution of 5% S-sulfonated keratin in a keratin solution of from 1: 0.1 to 1: 2 keratin: hydroxyapatite mass ratio. , Preferably by suspending 1: 1. Upon freezing and subsequent freeze drying, a homogeneous mixture of keratin and hydroxyapatite is produced with a porous spongy material suitable for use in bone graft products.

In another embodiment of the invention, the S-sulfonated keratin is reconstituted with a tough and dense material for use as a bone fixation product. This is accomplished by compacting keratin proteins. In general, the S-sulfonated keratin, which is ground to a particle size of 125 to 200 microns, is mixed with water in a ratio of 1: 0.1 to 1:10, preferably 1: 1 to aid in powder compaction. Hydroxyapatite is also incorporated into the mixture at levels of 0 to 60% by mass, preferably 0 to 10% by mass. The mixture was charged to a die and pressurized for a time ranging from 1 to 30 minutes at a pressure ranging from 10,000 to 50,000 kPa. Upon removal from the die, the cylinder is cut from the pellet and the cylinder is immersed in a chemical treatment solution to wash out the porogen and leave a porous material, while also removing sulfonate functionality from keratin and restoring disulfide crosslinking to the protein. Such chemical treatment may take one of two forms. First, the cylinder is treated for 2 to 48 hours, preferably 18 hours, using a solution of ammonium thioglycolate, preferably 0.25 M, containing 0.1 M sodium phosphate buffered to pH 7.0. can do. Residual chemicals are removed by subsequent washing of the pellets in water. After washing, the cylinder is lyophilized. Alternatively, the cylinder may be subjected to chemical treatment for 2 to 48 hours, preferably 18 hours in a solution containing thioglycolic acid, preferably 0.1 M. After a short wash in water, residual chemicals are removed by washing the cylinder in a buffer solution for 48 to 96 hours. Replenish the solution every 24 hours. After further washing in water, the cylinder is lyophilized.

The biological properties of the dense keratin materials have been demonstrated in vitro and the biophysical properties have been examined in vivo. In vitro, the material is not cytotoxic and supports the growth of human and sheep fibroblasts. Using both fibroblasts, direct contact of the porous material with the cells, as described in ISO 10993-5, produces the following effects. Wells of polystyrene cell culture plates with or without non-porous material as a control were initially seeded at about 10,000 cells (0 hour). During the first 24 hours after seeding, the cultures had a lag time as evidenced by a decrease in cell number. This phenomenon was recognized in all analyzes performed and a drop was observed in the control wells as well as those containing the test material. Experiments have shown that this lag time lasts less than 12 hours and exponential growth begins at this point. Population doubling occurs to sub-adhesion (approximately 80% adhesion) marking the end of the logarithmic growth phase approximately every 24 to 48 hours. This corresponds to the end of the experiment period (5 days or 120 hours). An extended period of experiment indicated a plateau in cell proliferation immediately after this with complete adherence of the culture. Contact inhibition and nutrient depletion play a major role in limiting the rate of proliferation at this point and monolayer cultures show signs of cell death (ie loss of membrane preservation, decrease in cell number, fear formation of individual cells). During the analysis, the cells were observed to attach to the upper surface of the disk. According to light microscopy, the morphological appearance of these cells was considered similar on all substrates compared to the non-material control. Similar analyzes with human fibroblasts showed very similar results with typical fibroblast proliferation curves occurring in the presence of porous materials, reaching approximately 80% adhesion after 120 hours of culture. Control wells reached 100% adhesion at this point.

Biophysical properties (elastic modulus, fracture modulus, tensile strength) were tested by subcutaneous implantation of materials into adult rats treated with thioglycolic acid and made from plates 12 mm long, 4 mm wide and 3 mm deep. Plates were removed from rats at 1, 3, 6 and 12 weeks and physical strength was evaluated. The modulus of elasticity showed a decrease of 40 to 70% over 3 to 6 weeks. The dry weight reduction in these two times was 5 to 10%, which is consistent with the studies done in sheep with a plate of porous material.

Another aspect of the invention is a tough and dense keratin material for use in bone fixation, made from heat compression of multiple layers of keratin films. Keratin films are readily prepared from S-sulfonated keratin using the method as described in WO 03/018673. Keratin films can also be made using a solution of S-sulfonated keratin containing a suspension of hydroxyapatite. Using this method, hydroxyapatite is incorporated into the keratin film at a level of 0-50% relative to the keratin mass. The resulting film can be layered and then compacted for a time ranging from 1 to 60 minutes at a temperature ranging from 50 to 200 ° C. at a pressure ranging from 10,000 to 100,000 kPa. After compaction, water is added to the compacted mixture and further compacted under the same conditions. The resulting material contains a homogeneous mixture of keratin and hydroxyapatite in the form of dense, tough blocks. The chemical treatment is then used to convert the S-sulfonate group to cystine. Under the same treatment and washing conditions as described above, thioglycolic acid or ammonium thioglycolate is used. Once dried, the resulting material can be machined into shapes useful as devices in orthopedic surgery such as screws or pins.

In an alternative approach, chemical treatment is applied to keratin films containing 0 to 60% of hydroxyapatite prior to stacking. Chemical treatment is accomplished by immersing the film in a solution of ammonium thioglycolate of a composition similar to that described above for 20 to 60 minutes. After washing the film several times in water to remove residual chemicals and drying, the resulting material contains keratin, in which the S-sulfo group is converted back to cystine. The film is then layered and compressed in the same manner as described above, the resulting material contains a homogeneous mixture of keratin and hydroxyapatite in the form of a dense, interstellar block. No additional chemical treatment is required, and the material can be machined into shapes useful as devices in orthopedic surgery such as screws or pins.

One aspect of the present invention is the use of a reducing agent such as ammonium thioglycolate or thioglycolic acid, as described above, to remove sulfonate functionality from proteins and restore disulfide bonds inherent in native keratin. In the sulfonate form, keratin is soluble above pH 4 and readily reabsorbed in vivo. In order to keep the material in the body for a long time and to control the rate of decomposition and resorption of the material, a reducing agent may be used to remove sulfonate functionality and crosslink the protein. The range in which the reducing agent is used, the exposure time and the concentration of the reagents affect the ratio of sulfonate group: disulfide bonds present in the material. This in turn affects strength and rate of degradation in vivo. Other crosslinkers such as those used to modify the properties of other biological biomaterials, such as glutaraldehyde and ethyldimethylaminopropylcarbodiimide hydrochloride (EDC), which are used to modify the properties of collagen biomaterials, are also It can be used to modify the properties of keratin biomaterials.

Example

Example 1a Porous Keratin Material

0.4 g of S-sulfonated keratin intermediate filament protein powder ground to a particle size of 125 to 300 microns is mixed with 0.5 ml of water and 0.024 g of sodium chloride and then left to stand for 5 minutes. The mixture is filled into a 12 mm diameter die and pressurized for 2 minutes at a pressure of 15,000 kPa. Upon removal from the die, a 3 mm diameter cylinder is cut from the pellet and the cylinder is immersed in a chemical treatment solution to wash out the porogen and leave a porous material, while also removing sulfonate functionality from keratin and disulfide crosslinking to the protein. Restore Such chemical treatment may take one of two forms. First, the cylinder can be treated for 18 hours using a solution of 0.25M ammonium thioglycolate containing 0.1M sodium phosphate buffered to pH 7.0. The residual chemical is removed by subsequent washing of the pellets in water for 10, 40 and 10 minutes in succession. After washing, the cylinder is lyophilized. Alternatively, the cylinder may be subjected to chemical treatment for 18 hours in a solution containing 0.1M thioglycolic acid. After a short wash in water, the residual chemicals are removed by washing the cylinder in a 0.1 M TRIS 11.25 mM calcium chloride solution for 72 hours. Replenish the solution every 24 hours. After further washing in water, the cylinder is lyophilized.

Example 1b Porous Keratin Material Containing Hydroxyapatite

The production of this material was the same as the method described in Example 1a, with the addition of 0.034 g hydroxyapatite at the same time as the inclusion of sodium chloride to produce the final product at the level of 6 mass% hydroxyapatite.

Example 1c Porous Keratin Material Containing Hydroxyapatite

To prepare porous keratin materials, a 5% keratin solution by suspending 0.5 g S-sulfonated wool keratin protein in water and then gradually adding 0.5 ml of 1M sodium hydroxide over vigorously stirred solution over approximately 2 hours. Was prepared. The pH of the solution was carefully monitored and increased to about pH 10 upon immediate addition of base, and then observed to drop gradually as the base was absorbed by dissolution of the protein. A final pH of 9.5 was obtained. The protein solution was centrifuged at 34,000 g to remove insoluble material and 0.5 g of hydroxyapatite was thoroughly mixed into the solution. The mixture was lyophilized and lyophilized to produce a porous material. The material was immersed in the same ammonium thioglycolate solution as used in Example 1a for 30 minutes, followed by 20 minutes of wash in 3 portions of water and freeze dried.

Example 2a Dense Keratin Material

0.5 g of S-sulfonated keratin protein powder ground to a particle size of 125 to 200 microns is mixed with 0.5 ml of water and then left to stand for 5 minutes. The mixture is filled into a 12 mm diameter die and pressurized for 2 minutes at a pressure of 15,000 kPa. Upon removal from the die, the desired shape, such as a 12 mm x 4 mm block, is cut from the pellets. The block is immersed in a chemical treatment solution to remove sulfonate functionality from keratin and restore disulfide crosslinking to the protein. Such chemical treatment may take one of two forms. First, a block can be treated for 18 hours using a solution of 0.25M ammonium thioglycolate containing 0.1M sodium phosphate buffered to pH 7.0. Residual chemicals are removed by subsequent washing of the blocks in water for 10, 40 and 10 minutes in succession. After washing, the blocks are dried in air at room temperature. Alternatively, the block may be subjected to chemical treatment for 18 hours in a solution containing 0.1M thioglycolic acid. After a short wash in water, the block is washed in a 0.1 M TRIS 11.25 mM calcium chloride solution for 48 hours to remove residual chemicals. Replenish the solution every 24 hours. After further washing in water, the blocks are dried at ambient temperature in air.

Example 2b Dense Keratin Material Containing Hydroxyapatite

The production of this material was the same as the method described in Example 2a, where 0.032 g hydroxyapatite was added to the keratin powder at the beginning of the process to produce a final product at the level of 6 mass% hydroxyapatite.

Example 2c: Density keratin materials made from keratin films

A 0.3 mm thick film of S-sulfonated keratin prepared by the method as outlined in WO 03/018673 was cut to 4 x 50 mm. Multiple films with a total mass of 1 g (corresponding to 50 films) are stacked in a heated steel block with an internal dimension of 5x50 mm, pressurized for 5 minutes at a pressure of 50,000 kPa and heated to 80 to 100 ° C through heating of the steel block. Kept at temperature. The formed keratin blocks were immersed in the ammonium thioglycolate solution described in Example 1a for 18 hours and subsequently washed in water for 10, 40 and 10 minutes. Air dry.

Alternatively, the film used contained 50% hydroxyapatite and was prepared as follows: 1.0 g S-sulfonated wool keratin protein was suspended in water. 0.2 g of glaserol and 1.0 ml of 1 M sodium hydroxide were gradually added to the vigorously stirred solution over approximately 2 hours. The pH of the solution was carefully monitored and increased to about pH 10 upon immediate addition of base, and then observed to drop gradually as the base was absorbed by dissolution of the protein. A final pH of 9.5 was obtained. The insoluble material was removed by centrifuging the protein solution at 27,000 g. 1.0 g of hydroxyapatite was pasted with a small amount of 99% ethanol (about 1 ml) and then thoroughly mixed in a 5% solution. The mixture was cast on 100 mm square Petri dishes and dried under ambient conditions.

Example 2d: Dense Keratin Material Made from Pretreated Keratin Films

A film of 0.3 mm thick S-sulfonated keratin prepared by the method as outlined in WO 03/018673 was treated for 30 minutes in the ammonium thioglycolate solution described in Example 1a. After treatment, the film was washed in water and dried to leave a film containing disulfide rather than an S-sulfonate functional group. These films were cut to 4 x 50 mm. Multiple films with a total mass of 1 g (corresponding to 70 films) are stacked in a heated steel block with an internal dimension of 5x50 mm, pressurized for 5 minutes at a pressure of 50,000 kPa and heated to 80 to 100 ° C by heating the steel block. Kept at temperature. The keratin blocks are removed, briefly immersed in water, pressurized on a heating block and compressed for 5 minutes at 20,000 kPa. The block is heated to 80-100 ° C. under pressure. Pressurized at 30,000 kpa for 30 minutes.

Alternatively, the film used contained 50% hydroxyapatite and was prepared as described for Example 2b. In the same manner as in Example 2c, a dense keratin material containing hydroxyapatite was prepared.

In the foregoing description, certain components or integrals of the present invention which are known to be equivalent are mentioned, and such equivalents are incorporated herein as described separately.

The invention will be useful in the medical field, especially in the field of bone injury. The keratin materials according to the present invention are biocompatible and can be used as bone substitutes and enhancement products.

The product can be used to replace lost bone, for example due to clinical symptoms such as trauma or tumors, and will promote healing by acting as a framework for laying the renal bone. The porous keratin backbone is reabsorbed and replaced by a new occlusal bone which will subsequently be remodeled into cavernous and cortical bone.

Claims (43)

Keratin-containing material for use in the preservation, repair and development of bone morphology and function. Porous keratin materials for use in bone replacement and augmentation. Dense keratin materials for use in bone fixation and passivation. The material of claim 1, wherein the keratin is S-sulfonated. The material of claim 1, wherein the keratin is concentrated in the intermediate filament protein. The keratin material according to claim 5, which is prepared by compacting solid keratin powder. 4. The dense material of claim 3, wherein the dense material is produced by compression of a keratin film. 8. Material according to any one of the preceding claims, containing up to 60% calcium salt. 8. Material according to claim 6 or 7, wherein the solid keratin is lyophilized after compression. Use of dense keratin materials in the manufacture of a medical support or skeleton in the preservation, repair and development of bone morphology and function. Use according to claim 10, wherein the keratin materials are S-sulfonated. Use according to claim 10 or 11, wherein the keratin is concentrated in the intermediate filament protein. a) compressing keratin in the presence of heat and water; b) strengthening the material; c) washing the material to remove residual chemicals; And d) drying the material, A method of forming a dense material of S-sulfonated keratin materials into orthopedic products. a) strengthening the keratin-containing starting material; b) washing the material to remove residual chemicals; c) drying the material; And d) compacting the keratin in the presence of heat and water, Method of forming dense material of S-sulfonated keratin as orthopedic product a) compacting keratin in the presence of soluble porogen; b) removing the porogen and strengthening the material; c) washing the protein material; And d) freeze drying the material; A method of forming a porous S-sulfonated enriched keratin material. The method of claim 15 wherein the porogen is selected from sodium chloride or another biocompatible salt, or glycerol or another biocompatible solvent. 17. The method according to claim 15 or 16, wherein the amount and type of porogen is adjusted to select pore size and infiltrate bone progenitor cells upon transplantation to promote colonization of keratin materials. 18. The method of any of claims 13 to 17, further comprising the addition of hydroxyapatite to the keratin starting material. 19. The method of any one of claims 13-18, wherein the keratin is concentrated in the intermediate filament protein. A keratin material prepared by the method of claim 13. A biocompatible material in the form of porous keratin that is concentrated in an intermediate filament protein for use in bone replacement / enhancement therapy. 22. The biocompatible material of claim 21 wherein the keratin is S-sulfonated. 23. The biocompatible material of claim 21 or 22 containing up to 60% calcium salt. The biocompatible material according to any one of claims 21 to 23, wherein the material is prepared by compacting solid keratin powder. The biocompatible material of claim 24 wherein the material is lyophilized after compression. 26. The biocompatible material according to any one of claims 21 to 25, wherein the material is made from a solution of keratin. 27. The biocompatible material of claim 26 wherein the solution of keratin is lyophilized. An orthopedic medical material made from biocompatible keratin materials for treating fractures by fixation and immobilization of internal fixation and bone fragments. The orthopedic material of claim 28, wherein the orthopedic material is made from an S-sulfonated keratin material. 30. The orthopedic material of claim 28 or 29, wherein the keratin materials are concentrated in intermediate filament proteins. The orthopedic material according to any one of claims 28 to 30, which is produced by compression of solid keratin powder. 31. The orthopedic material according to any one of claims 28 to 30, which is produced by compression of a keratin film. The orthopedic material according to any one of claims 28 to 30, which is made from a solution of keratin. The orthopedic material according to any one of claims 28 to 30, which contains up to 60% calcium salt. 34. The orthopedic material according to any one of claims 31 to 33, wherein the keratin is lyophilized after compression. 36. An orthopedic material according to any of claims 28 to 35, prepared according to the method of any one of claims 13 to 19. A method of reforming S-sulfonated keratin concentrated in an intermediate filament protein into a tough and dense biocompatible material for use as an internal fixation device for fracture treatment. 37. The method of claim 36, wherein the keratin is concentrated in the intermediate filament protein. 38. The method of claim 37 comprising compacting the biocompatible protein in the presence of moisture and chemicals. 40. The method of claim 39, wherein heat is also used to form the desired shape. 41. The method of any one of claims 37-40, which also involves the controlled use of a reducing agent to remove the sulfonate group from the S-sulfonated keratin and to reconstruct the disulfide originally present in the native keratin. 42. A biocompatible keratin concentrate material prepared according to any of claims 37-41. 29. The orthopedic material of claim 28, wherein the material is a plate, pin or screw.