JP2002291867A - Porous tissual skeleton forming material for repair or regeneration of tissue - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a biocompatible composite formed of a first fiber layer and an internally communicating continuous cell type three-dimensional porous foam, having a gradient in the composition and/or the microstructure in one or more directions and attached to the first fiber layer. SOLUTION: The foam can be formed of a mixture of bioabsorbable and biocompatible polymers, and the biocompatible composite formed of the polymers is suited especially for use in artificial processing of tissue, and can be formed so as to imitate a transition area or a boundary area in tissue.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

FIELD OF THE INVENTION The present invention relates generally to the field of tissue repair and regeneration. In particular, the present invention relates to porous biocompatible, bioabsorbable foams having a constant gradient in composition (gradual changes in morphology and function) and / or templates for tissue regeneration, repair or enhanced implantation And a microstructure acting as a microstructure.

[0002]

2. Description of the Prior Art It has been recognized that open cell porous foams are clearly effective for use in tissue repair and regeneration. In tissue repair, the use of amorphous, bio-acceptable foams as porous plugs to fill the pores of bone was initially intensively studied. Brekke et al. (U.S. Pat. No. 4,186,4)
No. 48) describes the use of a porous mesh plug composed of a polyhydroxy acid polymer such as polylactide to heal the pores of bone. In recent years, for example, US Pat.
Nos. 2,895 (Mikos) and 5,514,378
(Mikos et al.), US Patent No. 5,755 using vacuum foaming technology.
5,792 (Brekke) and 5,133,755
No. 5,716,413 (Walter et al.) And US Pat.
Nos. 07,474 (Athanasiou et al.) And U.S. Pat. Nos. 5,686,091 (Leong et al.) And 5,677,355 (Shalaby) which use polymer melts with labile compounds that sublime above room temperature. Et al.), U.S. Pat. No. 5,770,193 (Vacanti
Others, US Pat. No. 5,769,899 (Schwartz et al.), US Pat. No. 5,711,960 (Shikinami), and the like, have attempted to produce a TE skeleton forming material by different methods.
Also, Hinsch et al. (EPA 274,898) describe a range of about 10 μm to about 200 μm
A porous open-cell foam of a polyhydroxy acid having pores of the following size is described. The foam described by Hincsh can be reinforced with fibers, yarns, braids, knitted fibers, scrims and the like. The literature Hincsh also discloses various polyhydroxy acids and poly-L-lactide, poly-D
It describes the use of copolymers such as L-lactide, polyglycolide, and polydioxanone. This Hinc
Sh foams have the advantage of having a regular pore size and shape that can be controlled by process conditions, selected solvents, and additives.

[0003] However, the above technique has limitations in producing a skeleton-forming material having a certain gradient structure. Most scaffold-forming materials are isotropic in their morphology and function and lack anisotropic features in natural tissues.
Furthermore, it has been difficult in the prior art to create a three-dimensional skeleton-forming material capable of controlling the spatial distribution of various pore shapes. The reported method for producing microstructure controlled foams is a low temperature method and has many advantages over other conventional techniques. For example, the method can incorporate thermally sensitive compounds such as proteins, drugs, and other additives, including thermally and hydrolytically labile absorbent polymers.

More recently, Athanasiou et al. (US Pat. No. 5,607,474) have disclosed a two-layer foam for repairing osteochondral defects where two dissimilar tissues are present. Suggests the use of body devices. This Atha
The device of nasiou is composed of a first layer and a second layer which are partially separated and are integrally joined at a later stage. Each skeleton forming layer is configured to have rigidity and compressibility corresponding to cartilage and bone tissue, respectively. Since cartilage and bone often form adjacent layers in the body, this method is intended to more closely resemble human anatomy. However, the interface between cartilage and bone in the human body is not a discontinuous joint of two dissimilar materials with sharp changes in anatomical features and / or mechanical properties. That is,
Chondrocytes have a distinct cell morphology and orientation depending on the location of the chondrocytes relative to the underlying bone structure, and the differences in chondrocyte morphology and orientation cause the cartilage boundary of the underlying bone from the outer surface of the cartilage. A continuous transition structure up to the surface is formed. Thus, while Athanasiou's two-layer system has improved, it is not analogous to the tissue interface present in the human body.

Another technique for making three-dimensional laminate foams is described in Mikos et al. (US Pat. No. 5,51,51).
No. 4,378). In this tedious technique, a porous membrane is created by first drying a polymer solution containing elutable salt crystals. Thereafter, a three-dimensional structure is obtained by laminating several membranes together, which are cut to the desired contour.

One of the major drawbacks of the prior art relating to the three-dimensional porous skeleton forming material used for regeneration of living tissue such as cartilage is that the microstructure of the material is irregular. Such skeletal materials, unlike native tissues, have not changed in morphology or structure.
Moreover, such scaffolds do not provide nutrient and fluid transfer suitable for many applications. In addition, the laminate-like structure is not completely integrated and tends to peel under in vivo conditions.

[0007]

SUMMARY OF THE INVENTION It is therefore an object of the present invention to provide a continuous gradient of form, structure and / or material.
The purpose of the present invention is to provide a bio-acceptable, bio-absorbable foam having an adient). Furthermore, the foam used in the artificial treatment or repair or regeneration of tissue constitutes a template that organizes at the microstructural level, facilitating the infiltration, proliferation and differentiation of cells that occur in the regeneration of functional tissue. It is preferable to have a certain structure.

[0008]

SUMMARY OF THE INVENTION The present invention provides a composite comprising a first layer, which is a biocompatible filamentary layer, and a second layer of biocompatible foam. This composite structure makes it possible to create a structure with unique mechanical properties. This fiber layer allows the composite to have mechanical strength that can vary depending on the design, different bioabsorption profiles and different local environments for cell infiltration and inoculation, which are advantageous in a wide range of medical applications. is there.
The fibrous layers may be made from various biocompatible polymers and mixtures of biocompatible polymers, which are preferably bioabsorbable. The biocompatible foam may be either a foam with a different gradient structure or a foam with a flow path. The foam with the gradient structure has a generally continuous transition in at least one property selected from the group consisting of composition, stiffness, flexibility, bioabsorption rate, porous structure, and / or microstructure. I have. Foams having this gradient structure can be made with a mixture of absorbent polymers that form a compositional transition gradient from one polymer material to another. Also, if a single chemical composition can be adequately adapted for an application, the invention can be used in one or more directions that can resemble the anatomical features of tissue (eg, cartilage, skin, bone, etc.). A biocompatible foam having microstructural changes in structure is provided. The foam with the flow path has a flow path that extends through the foam to facilitate the movement of cells and the flow of nutrients throughout the foam with the flow path.

The present invention also provides a method for repairing or regenerating a tissue, the method comprising the steps of: providing a first tissue at a location in a complex having appropriate properties to promote tissue growth. Contacting the composite. This composite structure is particularly useful for tissue regeneration between two or more different tissues. In the simplest case of a multicellular system, one cell type may be on one side of the scaffold and another cell type may be on another side of the scaffold. . As an example of such playback,
(A) In the case of vascular tissue, smooth muscle is present on the outside and endothelial cells are present on the inside to regenerate the vasculature; By orienting the fibrous surface to the outside.

[0010]

DETAILED DESCRIPTION OF THE INVENTION The present invention discloses a novel microstructured bioabsorbable porous polymer foam. The properties of the foam can be adjusted to suit the desired application by selecting the appropriate conditions for forming the foam during lyophilization. The properties of this absorbent polymer have distinct advantages over the prior art, where the skeletal material is generally isotropic or irregular in structure. However, the foam used in the artificial processing of tissue (ie, repair or regeneration) organizes at the microstructural level, resulting in anatomical, biomechanical and biochemical properties in normal tissue. It is preferable to have a certain structure that constitutes a template that facilitates cell organization and tissue regeneration having Such foams can be used for the repair or regeneration of tissues (including organs) in animals such as domestic animals, primates and humans.

The properties of the foams of the present invention can be tailored to suit the desired application by selecting appropriate conditions for lyophilization to have one or more of the following properties. That is, (1) about 10 μm to about 2 μm forming a passage for cell internal growth and diffusion of nutrients.
(2) various porosity in the range of about 20% to about 98%, preferably about 80% to about 95%, (3) selection. Gradient in the size of pores along a certain direction corresponding to typical cell culture, (4) cell infiltration extending into the foam,
Channels for improving angiogenesis and nutrient diffusion,
(5) micropatterning of pores on the surface for cell organization; (6) selectivity of pore shape and / or orientation (eg, generally spherical, elliptical, cylindrical); (7) mechanical properties. Anisotropic, (8) composite foams with gradients of polymer composition that exhibit or have the advantage of different cellular responses to different materials; (9) different polymer compositions to form structures with portions that break at different rates Mixture, (10) RGD, growth factors (PDGF, TGF-
lyophilized or foam coated with a pharmaceutically active compound, including but not limited to biological factors such as β, VEGF, BMP, FGF, etc. (1
1) the ability to create three-dimensional shapes and devices with preferred microstructures, and (12) lyophilization with another part or medical device to form a composite structure. The tuned properties of these absorbent polymers are a distinct advantage over the prior art, where the scaffold-forming materials are generally isotropic or irregular structures that do not have a favorable morphology at the pore level. have. However,
The foam used in the tissue skeletal material has a certain structure that organizes at the microstructural level and forms a template that facilitates the organization of cells that can resemble natural tissue. preferable. Cells attach, proliferate and differentiate along or through the shape of this structure. This allows for the culturing of tissue that can resemble the anatomical characteristics of the actual tissue to a large extent.

For example, as shown in FIG. 3, the direction of the main axis of the pores can be changed from within the same plane as the foam to a direction perpendicular to the plane of the foam. Also, as can be seen from FIG. 3, the size of the pores can vary from about 30 μm to about 50 μm pores to about 100 μm to about 200 μm pores in a foam having a porous gradient structure. Desirably, the foam structure can be formed to facilitate the repair or regeneration of a human tissue joint, such as cartilage, to a bone joint present within the joint. The foam may be small (ie, about 30 μm to about 150 μm in diameter) round pores to relatively large columnar (ie, about 30 μm to about 400 μm, preferably about 100 μm to about 400 μm in diameter, and often in length. (The ratio of diameter to diameter is at least 2). FIG. 4 is a diagram showing a foam having a flow channel in FIGS. 2 and 3. These channels formed by the method of the present invention extend from the surface on one end side of the foam in the thickness direction of the foam. The length of this channel is generally at least twice the average diameter of the pores, preferably at least four times, and most preferably at least eight times.
It is twice. It should be noted that the channels for most applications are at least 200 microns long and may extend through the thickness of the foam. The diameter of the flow path is at least one time, preferably at least two to three times the average diameter of the pores. Of course, the size and diameter of the channel can be selected according to the desired function of the channel, such as a channel for cell infiltration, nutrient diffusion, or angiogenesis.

There are many biological tissues that exhibit gradient structures. That is, bone, spinal discs, articular cartilage,
Meniscus, fibrous cartilage, tendons, ligaments, dura, skin, vascular grafts, nerves, liver, pancreas and the like. The following examples illustrate some of the possible uses of the scaffolds with gradient structures. The construction of scaffolds that artificially manipulate tissue to facilitate the growth of this organ structure benefits greatly from the ability to form gradient structures in the scaffold.

[0014]cartilage  Articular cartilage forms joints in humans and animals
All bone ends. This cartilage distributes power
Mechanisms and supporting surfaces between different bones
Works. Without this articular cartilage, stress concentration and
Joints are not easily moved due to friction and friction. Also joints
Loss of cartilage generally leads to painful arthritis and joints
A decrease in the movement occurs. Fig. 10 Morphology of healthy cartilage
Various features are schematically illustrated.

[0015] Articular cartilage is a good example of a structure having a natural gradient. That is, articular cartilage is composed of four different regions, which include the superficial or peripheral region within the first 10% to 20% of the structure (this region includes the surface of the joint), 40% to 60% of the structure
, A deep region close to the reference line, and a transition region between bone and cartilage constituted by calcified cartilage. In addition, subchondral bone is present near the baseline and has transitioned to reticular bone. In the superficial or peripheral region, collagen fibrils are parallel to the surface. These fibers are oriented to resist the shear forces that occur during normal articulation. In the middle region, there is a tissue in which collagen fibers having a considerably large diameter are arranged irregularly. In addition, in the deeper regions, there are bundles of thicker collagen fibers that are perpendicular to the surface and penetrate into the calcified cartilage. These cells tend to cluster and arrange in a spheroidal form. In addition, the calcified cartilage region has small cells with relatively small cytoplasm.

A preferred embodiment of the present invention is directed to forming a foam structure having a gradient that can act as a template for a number of different regions. These foam structures can be formed into various shapes to regenerate or repair osteochondral defects and cartilage. One example of an effective foam structure can be a cylindrical shape having a rough dimension of 10 mm diameter and 10 mm length. In addition, the top can be about 1 mm thick and can be a low porous layer to control the passage of fluids. By using an appropriate treatment method, the porosity of the surface of the foam can be adjusted. That is, the porosity of the skin-like surface can vary from completely impermeable to completely porous.
Fluid permeability can be adjusted by the porosity of the surface. Under such a coating, the structure can be composed of three types of regions. The upper porous region is close to the cartilage tissue and the lower porous region is close to the bone tissue, with a transition region between the upper and lower regions. In the case of articular cartilage, it is presently preferred that the stiffness (elastic modulus) of the upper and lower porous layers be at least equivalent to the corresponding adjacent tissue at the time of implantation. That is, in such a case, these porous layers can support the environmental load and protect the infiltrating cells until they differentiate and combine to grow into a tissue that can support the load. For example, a porous structure used for the peripheral region of the superficial layer has elongated pores, and the orientation of the structure can be parallel to the surface of the host cartilage. However, the deep region is 100 μm
About 8 (about 80 μm to about 120 μm)
It has a porosity of 0% to about 95%. That is,
This area is expected to be infiltrated by chondrocytes. Further below this region may be a region having relatively large pores (about 100 μm to about 200 μm) and porosity ranging from about 50% to about 80%. Such one
A porous foam of from 00 μm to about 200 μm can have a structure in which the struts or walls of the pores are relatively large and perpendicular to the load, as well as a structure that can support the load with a natural structure. . In addition, large pores (about 150 μm to about 300 μm) with high rigidity that is structurally comparable to the reticulated bone at the bottom of the structure
is necessary. Therefore, the foam in this portion can be reinforced by fibers formed of ceramic particles, calcium phosphate, and the like.

[0017] Recent data from the present inventors' studies support the assumption that cell invasion can be regulated by stomata size. In these studies, a skeletal material made of 95/5 mol% poly ((L) lactide-co-ε-caprolactone) having pores of about 80 μm size (static) (Under conditions) showed a chondrocyte infiltration of about 30 cells / mm ² . Also, 40/60 mol% of poly (ε-caprolactone-) having pores having a size of about 100 μm.
The scaffold forming material (co- (L) lactide) showed a statistically significant difference (under static conditions) of a large cell infiltration of 50 cells / mm ² . In both of these cases, the cells were bovine chondrocytes. In addition, about 80
A very simple gradient structure with a pore size change from μm to about 150 μm provided a structure in which the area with relatively large pores was more easily infiltrated by chondrocytes. On the other hand, chondrocytes are not present in a region having relatively small pores, or a second cell type (eg, fibroblast)
Was filled by

In a compositionally graded foam, a mixture of two or more elastomeric copolymers or highly elastic semi-crystalline polymers and additives such as growth factors or granules is a preferred spatial The organization can first select the desired pore gradient to develop. The compositional gradients are then constructed primarily by differences in the polymer-solvent phase separation of each system, using the various techniques referred to in the preferred methods of making foams with gradient structures. Foam structures with such gradients show an effective response to chondrocytes or osteoblasts due to their spatial arrangement.

In addition, the purpose of the functional gradient is to distribute stress more evenly in regions where the mechanical and / or physical properties are changing to reduce the stress concentration effects of rapidly changing interfaces. . This condition is more similar to actual biological tissues and structures where the structural transitions between different tissues such as cartilage and bone are gradual. It is therefore an object of the present invention to provide an implant in which a functional gradient exists between the material phases. Thus, the present invention provides a multi-phase, functionally staged, bioabsorbable implant with attachment means for use in surgical repair of osteochondral defects or osteoarthritis. Several patents have proposed systems for repairing cartilage that can be used with the porous scaffolds of the present invention. For example, US Pat. No. 5,769,899 describes a device for repairing a cartilage defect, and US Pat. No. 5,713,899.
No. 374 describes a method of fixing a cartilage repair device with a bone anchor (both of which are incorporated herein by reference).

[0020]Bone  Gradient structures occur naturally at the bone / cartilage interface
You. In the study of the present inventors, the difference in material
It was found to have a significant effect. That is, the primary osteoblast
Cellular polymer film (95/5 (L) -lactide-co
-Glycolide copolymer, 90/10 glycolide-co-
(L) lactide copolymer, 95/5 (L) -lactide
-Co-ε-caprolactone copolymer, 75/25 gly
Colide-co- (L) lactide copolymer and 40/6
0ε-caprolactone-co- (L) lactide copolymer
-) And knitted mesh (95/5 (L) lactide-ko
-Glycolide and 90/10 glycolide-co- (L)
Lactide copolymers)
The answer was evaluated in vitro. From these results, 6 days of culture
Later on, osteoblasts were transformed into all biodegradable polymer films and
And grows well on the mesh
Was. 40 / 60ε-caprolactone-co- (L) lacti
Except for copolymer copolymer films,
Mer film is also compared to tissue culture polystyrene (control)
No significant promoting effect on the differentiation of primary rat osteoblasts
Was. On the other hand, 40 / 60ε-caprolactone-co-
Other films made with (L) lactide copolymer
Alkali film increased compared to film and TCPS
Sphatase activity and osteocalcin mRNA expression
The enhanced differentiation of cultured osteoblasts demonstrated by the present
Indicated. Therefore, different absorbent materials can be used to improve cell function and function.
It is evident that the differentiation and differentiation change significantly. Follow
To select the best material for cell growth and differentiation
More identical by using a composite material with a gradient structure of composition
Regeneration by different cell types in skeleton-forming material
Can be optimized.

Thus, in bone repair or regeneration devices or scaffolds, linear, branched or star-structured homopolymers or copolymers containing ε-caprolactone (random, block, segmented block, tapered) Block, graft, triblock, etc.)
The device prepared by the above method is particularly preferred. It is believed that aliphatic polyester copolymers containing ε-caprolactone in the range of about 30% to about 99% by weight are presently preferred. Suitable repeating units that can be copolymerized with ε-caprolactone are well known in the art. For example, suitable comonomers that can be copolymerized with ε-caprolactone include lactic acid, lactide (including L-, D-, meso and D, L mixtures), glycolic acid, glycolide,
p-dioxanone (1,4-dioxan-2-one),
Trimethylene carbonate (1,3-dioxane-2-
ON), δ-valerolactone, β-butyrolactone, ε
-Decalactone, 2,5-diketomorpholine, pivalolactone, α, α-diethylpropiolactone, ethylene carbonate, ethylene oxalate, 3-methyl-
1,4-dioxane-2,5-dione, 3,3-diethyl-1,4-dioxane-2,5-dione, γ-butyrolactone, 1,4-dioxepan-2-one, 1,5-
Dioxepan-2-one, 6,6-dimethyl-dioxepan-2-one, 6,8-dioxabicyclooctane-
7-one, and combinations thereof.

As a preferred medical device or tissue skeleton forming material for repairing and / or regenerating bone tissue containing a bioabsorbable polymer made of ε-caprolactone, a porous foam skeleton forming material (the present application) ), Fibrous three-dimensional drawn filaments, non-woven fabrics, woven fabrics, knitted or braided tissue scaffold-forming materials, composites containing reinforcing fibers, substrates, and mixtures thereof. Absent.

[0023]Skin  Another example of a tissue having a gradient structure is the skin.
You. The basic structure of the skin is two different types, but firm
It has layers that are integrated, and the thickness of each layer is
Different in different places. For example, the outer layer or epidermis
Avascular, small number of immune cells (Langerhans cells) and
Keratinocytes with melanocytes
It is mainly composed. This keratinocyte is keratin
Generates fiber and corneosite envelopes, which
Provide durability and protective functionality to the epidermis. These structures
Development is completely dependent on the epidermal differentiation state. Sand
The epidermis changes as cells move further away from the basement membrane
Form epithelium stratified by expression pattern. This differentiation
The stratified layer of expressing cells is shaped to maintain epidermal function.
Need to be implemented. Under the epidermis is the dermis,
The skin is a dense, irregular connective tissue with many blood vessels. this
The layers are highly dense with collagenous and elastic fibers
Thus, these fibers provide this layer with exceptional elasticity and strength.
Grant. Fibroblasts are the major cell type in this layer.
You. In addition, there is a basement membrane between these two layers,
Membranes act as attachment sites for epidermal cells, and these cells
Acts to regulate the function and differentiation of On the basement membrane
The layer of keratinocytes directly attached has the shape of a cube
And highly aligned. This adhesion state and structure
Is critical for producing highly scaly structures in the epidermis.
It becomes a necessary condition. In other words, this basal layer repairs the epidermis and
And a source of precursor cells for replacement. Ma
The scaly layer provides strength and resistance to injury and infection
Gives the property.

It should be considered that any material used for skin replacement can induce the infiltration of cells, such as fibroblasts, necessary to produce skin components in the healed tissue. In addition, it should be appreciated that the material preferably enhances, rather than inhibits, the rate of re-epithelialization in a manner such that another basal layer of the epidermis is formed. Materials that allow infiltration into the scaffold by moving keratinocytes can produce partially differentiated cells.

For this reason, the porous structure which facilitates the regulation of the access of specific cell types and the regeneration of natural tissues can have functional advantages. FIG. 11 (A), FIG.
(B) and FIG. 12 are diagrams respectively showing the microstructure of the foam skeleton forming material of the present invention. Further, FIG.
(× 100 magnification) and FIG. 14 (40 × composite photograph) show photographic evidence of infiltration of fibroblasts, macrophages, macrophage giant cells and endothelial-like cells into 0.5 mm foam. The foam tissue skeleton-forming material 101 shown in both of these photographs has ε-caprolactone-
Co-glycolide copolymer and ε-caprolactone
A 50:50 mixture of the co-lactide copolymer (made as described in Example 7). In addition, these photographs are 1.5 cm x 1.
Images were taken 8 days after implantation into a 5 cm x 0.2 cm resected wound model. Both pictures clearly show the complete incorporation of the substrate into the granulation tissue bed. Also, the dense fibrous tissue above the foam tissue scaffolding material appears to provide a suitable substrate for overgrowth of the epidermis. In the foam tissue skeleton forming material shown in FIG.
F is further incorporated. That is, it can be clearly seen that a growth factor such as PDGF is required in a wound healing model in which a wound has been formed.

According to the inventor's initial research, about 150
μm to about 3 mm, preferably about 300 μm to about 15
00 μm, most preferably from about 500 μm to about 1000
It would be desirable to use a foam tissue scaffold having a thickness of μm as the skin scaffold. Distinctly different skin injuries (ie, diabetic ulcers, venous stasis ulcers, pressure ulcers, burns, etc.) require different foam thicknesses. In addition, the condition of the patient may require the incorporation of growth factors, antibiotics and antimicrobial agents to facilitate wound healing.

[0027]Vascular graft  The formation of a tubular structure with a gradient is also relevant to the invention.
Has relatively large pores on the outside diameter and the size is the inside diameter
Or small transitions
Vessel graft with tubing in reverse condition is vascular tissue
In culture of endothelial cells and smooth muscle cells for culture
Useful.

The multi-layered tubular structure allows tissue regeneration similar to the mechanical and / or biological properties of blood vessels and can be used as a vascular graft. That is, a plurality of concentric layers formed with different compositions under different processing conditions can provide desired mechanical properties, bioabsorbability, and tissue ingrowth rate. The innermost luminal layer is optimized for endothelialization by adjusting its surface porosity and possible additions to the surface treatment. On the other hand, the outermost adventitia layer of the vascular graft optimizes its porosity (porosity, pore size, pore shape and pore size distribution) to provide a bioactive agent, drug, or It is adjusted to induce tissue ingrowth by incorporating cells. In this case, a low porosity barrier layer may or may not be provided between these two types of porous layers in order to increase strength and reduce leakage.

[0029]Foam composition  Various absorbent polymers can be used to make the foam. Use
Examples of bio-acceptable and bio-absorbable polymers suitable for
And aliphatic polyester, poly (amino acid), copoly
(Ether-ester), polyalkylene oxalate
, Polyamide, poly (iminocarbonate), polio
Rutoester, polyoxaester, polyamide esthetic
Polyamine, amine-containing polyoxaester, poly (acid
Anhydrides), polyphosphazenes, biomolecules, and these
A polymer selected from the group consisting of a mixture of
You. Further, aliphatic polyesters for the purpose of the present invention and
Lactide (lactic acid, D-, L-, and meso-lactide)
Glycolide (including glycolic acid), ε-
Caprolactone, p-dioxanone (1,4-dioxa
-2-one), trimethylene carbonate (1,3-
Dioxan-2-one), trimethylene carbonate
Alkyl derivative, δ-valerolactone, β-butyrolact
Ton, γ-butyrolactone, ε-decalactone, hydro
Xybutyrate (repeating unit), hydroxyvalerate
(Repeating unit), 1,4-dioxepan-2-one (the
Dimeric 1,5,8,12-tetraoxacyclotetra
Decane-7,14-dione), 1,5-dioxepane-
2-one, 6,6-dimethyl-1,4-dioxane-2
-One, 2,5-diketomorpholine, pivalolactone,
α, α-diethylpropiolactone, ethylene carbonate
, Ethylene oxalate, 3-methyl-1,4-di
Oxane-2,5-dione, 3,3-diethyl-1,4
-Dioxane-2,5-dione, 6,8-dioxabishi
Clooctane-7-one, and their polymer blends
Product homopolymers and copolymers
Not limited to In addition, the poly (iminoka) according to the object of the present invention
Literature)Handbook of Biodegradabl
e Polymers(Edited by Domb, Kost and Wisemen, Hard
wood Academic Press, 1997, pp. 251 to 2
72) by Kemnitzer and Kohn.
Is included. Copoly (ether-ether) for the purposes of the present invention
Esters), as described by Cohn and Younes
(“Journal of Biomaterials Research”, Vol. 22,
993 to 1009, 1988) and Cohn
Literature (Polymer Preprints (ACS Division of Polymer C
hemistry) Vol. 30 (1), p. 498, 1989)
The copolyester-ether (e.g., PEO /
PLA). Polyalkylene for the purpose of the present invention
Oxalate is included herein as a reference.
U.S. Pat. Nos. 4,208,511 and 4,14
No. 1,087, No. 4,130,639, No. 4-1
Nos. 40,678, 4,105,034 and
No. 4,205,399. Sa
In addition, polyphosphazenes and L-lactide, D, L
-Lactide, lactic acid, glycolide, glycolic acid, para-
Dioxanone, trimethylene carbonate and ε-ca
Copolymers made with prolactone, etc., terpoly
And higher-order mixed monomer-based poly
The document Allcock is incorporated herein by reference.
(The Encyclopedia of Polymer Science, Vol. 13,
31-41, Wiley Intersciences, John Wile
y & Sons, 1988), and Vandorpe, Schacht, De
jardin and Lemmouchi (Handbook of Biodegradable
Polymers, edited by Domb, Kost and Wisemen, Hardwo
ok Academic Press, 1977, pp. 161 to 1
82). Also, with poly (anhydride)
The chemical formula HOOC-C wherein m is an integer in the range of 2 to 8
₆H_Four-O- (CH_Two)_m-O-C₆H_Four-COOH diacid and this compound
And aliphatic alpha-omega 2 of up to 12 carbon atoms
Includes those made by copolymers with acids. Sa
In addition, polyoxaesters, polyoxaamides and
Polyoxae containing a min group and / or an amide group
U.S. Pat.
No. 5,464,929, No. 5,595,751, No.
Nos. 5,597,579 and 5,607,687,
Nos. 5,618,552 and 5,620,698
No. 5,645,850 and 5,648,08
No. 8, No. 5,698,213, No. 5,700,5
No. 83 and No. 5,859, 150
It is listed. Polyorthoesters are referenced herein
Literature Heller (Handbook of Biodegrada
ble Polymers, edited by Domb, Kost and Wisemen, Ha
rdwook Academic Press, 1977, pp. 99-1
18).

In general, aliphatic polyesters are absorbent polymers and are suitable for making foams having a gradient structure. The aliphatic polyester can be a homopolymer or copolymer having a linear, branched or star structure (random, block, segmented block, tapered block, graft, triblock, etc.), but the linear copolymer is preferable. Suitable monomers for making such aliphatic homopolymers and copolymers are lactic acid, lactide (including D-, L-, meso, D, L mixtures), glycolic acid, glycolide, ε-caprolactone, p-dioxanone ( 1,4-dioxane-2-
On), trimethylene carbonate (1,3-dioxan-2-one), δ-valerolactone, β-butyrolactone, ε-decalactone, 2,5-diketomorpholine,
Pivalolactone, α, α-diethylpropiolactone,
Ethylene carbonate, ethylene oxalate, 3-methyl-1,4-dioxane-2,5-dione, 3,3-
Diethyl-1,4-dioxane-2,5-dione, γ-
Butyrolactone, 1,4-dioxepan-2-one,
1,5-dioxepan-2-one, 6,6-dimethyl-
It can be selected from, but not limited to, the group consisting of dioxepan-2-one, 6,8-dioxabicyclooctane-7-one, and combinations thereof.

[0031] Elastomer copolymers are also particularly useful in the present invention. Suitable bioabsorbable and biocompatible elastomers include elastomeric copolymers of ε-caprolactone and glycolide (preferably about 3
5:65 to about 65:35, more preferably 45: 5
A molar ratio of ε-caprolactone to glycolide of 5 to 35:65), ε-caprolactone and L-
Lactide, a D-lactide mixture of L-lactide or an elastomeric copolymer of lactide, including a lactic acid copolymer (preferably from about 35:65 to about 65:35, more preferably from 45:55 to 30:70 or about 95: 5
Having a molar ratio of ε-caprolactone to lactide of about 85:15), p-dioxanone (1,4-dioxan-2-one) and lactide elastomers including L-lactide, D-lactide and lactic acid Copolymers (preferably having a molar ratio of p-dioxanone to lactide of about 40:60 to about 60:40), elastomeric copolymers of ε-caprolactone and p-dioxanone (preferably about 30:70 to about 60:40). 70:30
Having a molar ratio of ε-caprolactone to p-dioxanone), an elastomeric copolymer of p-dioxanone and trimethylene carbonate (preferably about 3
An elastomeric copolymer of p-dioxanone to trimethylene carbonate from 0:70 to about 70:30, trimethylene carbonate and glycolide (preferably from about 30:70 to about 70:30 trimethylene) Elastomers having a molar ratio of carbonate to glycolide), trimethylene carbonate and lactide, including L-lactide, D-lactide, mixtures thereof, and lactic acid copolymers (preferably from about 30:70 to about 70:30). , And a molar ratio of trimethylene carbonate to lactide), and mixtures thereof.

[0032] Also, examples of suitable bioabsorbable elastomers are disclosed in US Pat.
Nos. 45,418, 4,057,537 and 5,468,253. These elastomeric polymers are available in 0.1 g / deciliter (g) in hexafluoroisopropanol (HFIP).
/ DL) about 1 when measured at 25 ° C in solution.
2 dL / g to about 4 dL / g, preferably about 1.2 dL
/ G to about 2 dL / g, most preferably about 1.4 dL / g.
g to about 2 dL / g.

[0033] Preferably, these elastomers exhibit high elongation and low modulus and have good tensile strength and good recovery properties. In a preferred embodiment of the present invention, the elastomer forming the foam comprises about 20
It exhibits a draw ratio of 0% or more, preferably about 500% or more.
These properties indicate the degree of elasticity of the bioabsorbable elastomer described above and are approximately 500 psi (35.1 kgf / cm).
² ) Above, preferably about 1000 psi (70.3 kg
Weight / cm ² ) or more, and about 50 lbs /
Inches (8.9 kgf / cm) or more, preferably about 80
Achieved while maintaining a tear strength of lbs / inch (14 kgf / cm) or more.

Suitable polymers or copolymers for forming foams with a gradient structure for tissue regeneration depend on several factors. That is, the chemical composition, the spatial distribution of the components, the molecular weight of the polymer, and the degree of crystallization, all determine in part the in vitro and in vivo behavior of the polymer. However, the choice of polymer to create a foam having a gradient structure for tissue regeneration is highly dependent on the following factors, including but not limited to: (B) mechanical properties in vivo; (c) cell responsiveness to materials for cell attachment, proliferation, migration and differentiation; and (d) biopermissivity. .

The nature of the material matrix that is occasionally absorbed into the body environment is important. However, differentiation in absorption time under in vivo conditions is also a criterion for mixing two different copolymers. For example, 35:65 ε-
A copolymer of caprolactone and glycolide (a relatively fast absorbing polymer) can be mixed with a 40:60 ε-caprolactone and (L) lactide copolymer (a relatively slow absorbing polymer) to form a foam.
Such a foam may have several different physical structures depending on the processing technique used. That is, whether these two components are two continuous layers interconnected irregularly, have a constant gradient structure in the thickness direction, or form an integral boundary between the two component layers. A laminate-type composite having portions can be formed. further,
The microstructure of these foams can be optimized to regenerate or repair the desired anatomy of the tissue undergoing the artificial treatment.

A preferred embodiment of the present invention is directed to using a polymer mixture to form a structure that transitions from one composition to another in a gradient manner. Foams having such a gradient structure are used for cartilage (joints, meniscus,
It is particularly advantageous in artificial treatment of tissue to repair or regenerate the structure of natural tissues such as septum, trachea, etc., esophagus, skin, bone and vascular tissue. For example, by mixing ε-caprolactone-co-lactide with an ε-caprolactone-co-glycolide elastomer (at a molar ratio of about 5:95), a soft sponge-like foam resembling the cartilage to bone transition The foam can be formed to have a transition from to a rigid foam. Of course, similar gradient effects or different absorption profiles,
Different polymer mixtures can be used to construct stress response profiles, or different gradient structures such as different degrees of elasticity. In addition, these foams may use the specific skeletal materials of the present invention, including but not limited to spinal discs, dura mater, nervous tissue, liver, pancreas, kidney, bladder, tendon, ligament and breast tissue. Can be used for organ replacement or regeneration.

The above elastomer polymer is freeze-dried,
Supercritical solvent foaming (ie, a method as described in EP 464,163 B1), gas injection extrusion, gas injection molding or extractable materials (ie, any salt known to those skilled in the art, such as salts, sugars, etc.). The foaming can be performed by a casting process involving (means). At present,
It may be preferable to produce a bioabsorbable, bioacceptable elastomeric foam by lyophilization. Suitable methods for lyophilizing elastomeric polymers to form foams are described in the Examples below and assigned to Ethicon, Inc., filed June 30, 1999, incorporated herein by reference. No. ETH-1352 "Process for Manufacture of Biomedical Foams"
facturing Bopmedical Foams).

The foams made in the present invention can be made by a modified polymer-solvent phase separation technique of the prior art which unexpectedly formed a gradient structure in the foam structure. Generally, the polymer solution comprises (a) heat-induced gelation / crystallization,
(B) solvent-free guided separation of solvent and polymer phases;
The two phases can be separated by any of four techniques: (c) chemically induced phase separation, and (d) thermally induced spinodal decomposition. The polymer solution is separated in a prepared manner into two separate phases or two continuous phases. Subsequent removal of the solvent phase results in a porous structure having pores in the micrometer range with a lower density than the original polymer (A.
T. Young, Microcellular Foam by Phase Separation (Microcellu
lar foams via phase separation) "(J. Vac. Sci. T
echnol. A4 (3), May / June 1986). The steps involved in making these foams include selecting the right solvent for the polymer that needs to be lyophilized and making a uniform solution. Next, the polymer solution is frozen and subjected to a vacuum drying step. The phase separation of the polymer solution is performed by this freezing step, and the solvent is sublimated and removed in the vacuum drying step, and the remaining porous polymer structure, that is, the porous foam of the internally communicating cells is dried.

Solvents generally suitable as starting points for selecting a solvent for the preferred absorbent aliphatic polyester include formic acid, ethyl formate, acetic acid, hexafluoroisopropanol (HFIP), cyclic ethers (ie, THF, D
MF and PDO), acetone, acetates of C2 to C5 alcohols (such as ethyl acetate and t-butyl acetate), glyme (ie, monoglyme, ethyl glyme, diglyme, ethyl diglyme, triglyme, butyl diglyme and Tetraglyme), methyl ethyl ketone, dipropylene glycol methyl ether, lactone (γ-valerolactone, δ
Valerolactone, β-butyrolactone, γ-butyrolactone, etc.), 1,4-dioxane, 1,3-dioxolan, 1,3-dioxolan-2-one (ethylene carbonate), dimethyl carbonate, benzene, toluene, benzyl alcohol, p-xylene, naphthalene,
Including a solvent selected from the group consisting of tetrahydrofuran, N-methylpyrrolidone, dimethylformamide, chloroform, 1,2-dichloromethane, morpholine, dimethylsulfoxide, hexafluoroacetone sesquihydrate (HFAS), anisole, and mixtures thereof. But not limited to these. Among these solvents, 1,4-dioxane is a preferred solvent. In addition,
A homogeneous solution of the polymer in this solution can be made by standard techniques.

Thus, it will be appreciated that the applicable polymer concentration or amount of solvent available will vary from system to system. Suitable phase diagram curves for some systems are already known. However, if a suitable curve is not available, this curve can be created by known techniques. For example, one example of such a suitable technique is the literature Smolders, Van Aartsen and Steenbe.
rgen (Kolloid-Z, uZ Polymere, 243, 14 (1971
Year)). For general use, the amount of polymer in the solution can vary from about 0.5% to about 90% by weight, preferably from 0.5% to about 30% by weight, and the polymer in any solvent And the final properties of the desired foam.

In addition, solid materials can be added to the polymer-solvent system. The solid material added to the polymer-solvent system preferably does not react with the polymer or solvent. Suitable solid materials include materials that promote tissue regeneration or regrowth, buffers, reinforcements or porous modifiers, and the like. Preferred solid materials include demineralized bone particles, calcium phosphate particles, or calcium carbonate particles for bone repair,
Examples include, but are not limited to, dissolvable solid materials for pore formation, bioabsorbable polymer particles that do not dissolve in the solvent system as the reinforcing material, or polymer particles that form pores when absorbed.

Suitable elutable solid materials include salts (ie, sodium chloride, potassium chloride, calcium chloride,
Sodium tartrate, sodium citrate, etc.), biocompatible mono- and disaccharides (ie, glucose, fructose, dextrose, maltose, lactose and sucrose), polysaccharides (ie, starch, alginate), water-soluble proteins (ie, gelatin) And agarose) and non-toxic elutable materials selected from the group consisting of: Generally, these materials are all less than about 1 mm, preferably from about 50 μm to 5 μm.
It has an average diameter of 00 μm. Also, these particles make up about 1% to about 50% by volume of the total volume of the particles and the polymer-solvent mixture, where the total volume% is equal to 100% by volume. The dissolvable material, along with the dissolvable material, is immersed in a solvent that dissolves the particles in an amount of time that elutes substantially all of the particles but does not dissolve the foam or change disadvantageously. Can be removed. The preferred extraction solvent is water, with distilled deionized water being most preferred. This process is described in U.S. Patent No. 5,514,378, which is incorporated herein by reference. Preferably, unless accelerated absorption of the foam is desired, the foam is dried at low temperature and / or vacuum after completion of the dissolution process to minimize its hydrolysis.

After forming the polymer-solvent mixture, the mixture is solidified. For a particular polymer-solvent system, the freezing point, melting temperature, and apparent glass transition point of the polymer-solvent system can be determined by standard differential calorimetry (DSC) techniques. In theory, but not meant to limit the scope of the invention, it is believed that upon cooling of the polymer-solvent system, initial solidification occurs at a temperature near or below the freezing point of the solvent. This corresponds to the solidification of most of the solvent in the system. This initial solidification appears as a first exothermic peak. Thereafter, a second freezing point occurs as the solvent remaining with the polymer solidifies. This second freezing point is indicated as a second exothermic peak. In addition, a clear Tg is exhibited at temperatures where the completely frozen system exhibits a first exothermic shift upon reheating.

An important parameter to be adjusted is the polymer
The freezing rate of the solvent system. That is, the type of pore morphology fixed during the freezing process is a function of the thermodynamics of the solution, the rate of freezing, the temperature at which it is cooled, the concentration of the solution, and the nucleation of a homogeneous or heterogeneous system. A detailed description of such phase separation phenomena is incorporated herein by reference (AT Young, "Microcellular Foams by Phase Separation" (J.
Vac. Sci. Technol. A4 (3), May / June 1986)
And S. Matsuda, “Thermodynamics of Formation of Porous Polymer Film from Solution.
Polymeric Membrane from Solutions ”(Polymer J.
Vol. 23, No. 5, 435 to 444, 1991
Year).

Such previously reported polymer solutions may be coagulated in various ways, such as by placing or pouring the solution into a mold and cooling the mold in a suitable bath or on a freezer shelf. it can. Alternatively, the polymer solution can be sprayed by a sprayer and sprayed onto a cooled surface to solidify the sprayed film one by one. The cooled surface can be a medical device or a component or film thereof. Also, the shape of the solidified spray film will be similar to the shape of the sprayed surface. Alternatively, the coagulated mixture can be formed into a shape during cutting or freezing. Using these and other methods, foams can be formed into various shapes and sizes (ie, tubular, branched tubular, spherical, hemispherical, three-dimensional polygonal, elliptical (ie, kidney-shaped), Toroids, cones, truncated cones, pyramids, solid and hollow, and combinations thereof).

Alternatively, another method for making foamed parts is to use cold fingers (metal parts whose surface indicates the inside of the part to be manufactured). The cold finger is removed after immersion in the polymer in a suitable solvent. This process is very similar to dipping a stick of ice cream into warm chocolate to solidify and harden it, or dipping a molded body into rubber latex to form gloves and condoms. At this time,
The thickness and morphology of the foam produced is a function of the temperature, residence time and withdrawal speed of the cold finger in the mixture. That is, the longer the residence time and the lower the drawing speed, the thicker the coating. After withdrawal, the cryogenic finger is placed on a large heat capacity device in contact with the freeze-drying freezing tray. From this point, the primary and secondary drying processes as described above begin. The method is suitable for making tubes, bifurcated tubular structures or sleeves that can be formed into a shape that conforms to a portion of the device or animal anatomy.

In addition, the polymer solution is a film,
Solidification can be achieved by various inserts incorporated with the solution, such as scrims, wovens, nonwovens, knits or braided fibrous structures. In addition, this solution can be used for orthopedic implants (eg, screws, pins, nails and plates)
Or it can be made in conjunction with another structure, such as a tubular or branched tubular structure (a scaffold for vascular or tubular organs). These inserts can be formed from at least one biocompatible material and can be non-absorbable, absorbable, or a combination thereof.

The above polymer solution in the mold is cooled in a certain direction through the wall of the mold in contact with the freeze-drying shelf, and is processed in a certain heat cycle. The mold and its surface can be formed of any material that does not affect the polymer-solvent system, but is preferably formed of a material with high thermal conductivity. That is, heat transfer is transferred from the freeze dryer shelf through the mold wall into the polymer solution. Thereafter, when the temperature of the mixture falls below its gel point and / or freezing point, the mixture undergoes phase separation.

The form of this phase-separated system is fixed during the coagulation step of the freeze-drying process, and the formation of continuous pores begins with the start of the vacuum drying process and the solvent sublimates. However, the mixture in the container or mold cooled by the cooling radiator solidifies before completely freezing.
That is, even though the mixture appears to be in a solid state, there is initially some residual solvent associated with the uncrystallized polymer. In theory, but not in a sense to limit the invention, the freezing point is moved through the mixture by the cooling radiator and the solidification reaction is completed after the mixture has apparently solidified. Therefore, at any given time, the material ahead of the front of this freezing point is not as cold as the material behind it and is not completely frozen.

The present inventor has found that by applying a vacuum to the apparently solid polymer solvent mixture immediately after it has appeared in a solidified state, a foam having a gradient structure with various pore sizes and structures and channels is provided. Was found to be able to form. Therefore, the start time of the sublimation process (by vacuum treatment, ie, vacuum drying) is an important step in the method for forming a transition state in the structure. The start time of the sublimation process is affected by the thickness of the foam to be formed, the concentration of the solution, the heat transfer rate, and the direction of the heat transfer. It should be noted that those skilled in the art can respond to a particular polymer-solvent system by monitoring the thermal transition rate of the foam at various depths and locations within the foaming device using a thermocouple. It can be seen that this process can be monitored and characterized. That is, by controlling the sublimation process, a structure having a gradient and anisotropy in the form of pores can be formed. In addition, this method allows for the formation of microstructures similar to tissues such as cartilage, bone and skin. For example, in general,
When a vacuum is supplied immediately after the solution apparently solidifies, a flow path can be formed. However, when the same solution is further coagulated, the foam has relatively large pores near the surface to which the vacuum is supplied (opposite to the cooling radiator), and the foam on the side closer to the cooling radiator. It has relatively small pores in the area.

This treatment method is in contrast to prior art treatment methods which aim to completely freeze the solution to form a foam having a uniform microstructure (pores interconnecting irregularly). is there. In such prior art, the vacuum drying process was only performed after a considerable amount of time to complete the desired phase separation (U.S. Pat. Nos. 5,755,792 (Brekke) and 5,133,133). No. 755 (Brekke), No. 5,716,413 (Walter et al.), No. 5,60
7,474 (Athanasiou et al.), 5,686,09
No. 1 (Leong et al.) And No. 5,677,355 (Shalaby
Et al.) And European Patent Publication E0274898 (Hinsc
h) and EPA 594148 (Totakura)).

FIGS. 2, 3 and 4 show foams having various structures. For example, as shown in FIG. 3, the orientation of the major axis of the pores can vary from the same plane as the foam to being perpendicular to the plane of the foam. In theory, but not in a sense to limit the scope of the invention, in conventional foaming treatments, when the solvent crystallizes, the freezing front moves within the solution, causing the solution to crystallize parallel to the freezing front. It is thought to solidify within. However, if a vacuum is applied before the solution is completely frozen, the foam morphology is formed such that the pores are arranged generally parallel to the vacuum source, as shown in FIG.

As can be seen from FIG. 3, the size of the pores can vary from a small pore of about 10 μm to about 60 μm to a relatively large pore of about 60 μm to about 200 μm to a porous gradient foam. In this case, too, this state is obtained by supplying a vacuum to the apparently solidified solution before the solution has completely solidified. When adjusting the bubble size, the polymer concentration in the solution and the cooling rate are also important parameters. Preferably, the foam structure is formed so that it can act as a template for restoring a human tissue joint, such as cartilage, to a bone joint present in the joint. This foaming proceeds from small spherical pores to relatively large cylindrical (ie, having a diameter to length ratio of at least 2: 1). Further, the stiffness of the foam can be adjusted by mixing the foam structure or two different polymers having different Young's moduli.

The foam may have a flow path as shown in FIG. The channels formed by this method can traverse the thickness of the foam and generally range from about 30 to about 2
It has a diameter of 00 μm. Generally, the channels have a length that is at least twice, preferably at least four times, and most preferably at least eight times the average diameter of the channels. Of course, the size and diameter of the channel can be selected based on the desired functionality of the channel, such as channels for cell infiltration, nutrient diffusion or angiogenesis.

A person skilled in the art can set the above-mentioned directionality to an arbitrary three-dimensional state by setting a suitable mold configuration and, if necessary, changing the temperature of the wall of the mold. You can easily see what you can do. The following types of gradient structures can be formed by changes in pore size and / or shape through thickness having a uniform composition. That is, a structure in which there is one type and size of pores for a specific thickness, followed by another type and size of pores, a composition gradient mainly due to one type of composition on one side There is a composition gradient on the other side with another type of composition, a composition transition from one side to the other, a low-porosity thick film with a small-sized pore layer. This is followed by a structure with large pore areas, and a foam with vertical pores with spatial organization, these vertical pores can act as channels for nutrient diffusion. A structure in which these pores are created in a three-dimensional mold to form a three-dimensional foam having the desired microstructure combination of compositional and structural gradients. Generally, the foam formed in the container or mold has a thickness ranging from about 0.25 mm to about 100 mm, preferably from about 0.5 mm to about 50 mm. Thicker foams can be formed, but the lyophilization process takes significantly longer, making it difficult to control the final foam structure and increasing the residual solvent content.

As previously described, the process of the present invention for producing biocompatible foams involves temperatures above the apparent glass transition temperature and below the solidification temperature of the mixture (preferably just below the solidification temperature). It can be significantly reduced by performing the sublimation step under the conditions. That is, the time of the combined processing steps (freezing processing + primary drying processing + secondary drying processing) is much shorter than the time described in the prior art. For example, the combined processing time for aliphatic polyesters with volatile solvents is typically 72
The time is not more than 48 hours, preferably not more than 48 hours, more preferably not more than 24 hours, most preferably not more than 10 hours. In practice, this combined processing time is less than 3 hours for foams less than 1 mm thick, less than 6 hours for foams about 2 mm thick, and about 3 mm thick for foams of a particular aliphatic polyester. Less than 9 hours in body. On the other hand, in the prior art, this processing time is generally 72 hours or more. Further, the residual solvent concentration in the foam produced by the method of the present invention is extremely low. As described for aliphatic polyester foams made using 1,4-dioxane as the solvent, the concentration of residual 1,4-dioxane is 10 ppm (parts per million) or less, preferably 1 ppm or less, most preferably It was less than 100 ppb (parts pavilion).

It should be noted that those skilled in the art will be able to make the above-mentioned orientation into an arbitrary three-dimensional state by changing the temperature of the wall of the mold and the structure of the appropriate mold if necessary. Can be easily visually recognized.
According to the present invention, the following types of gradient structures can be created. That is, 1. 1. a structure in which the size and / or shape of the pores change through a certain thickness with a uniform composition; 2. a structure in which there is one type and size of pores for a particular thickness, followed by another type and size of pores; 3. a structure in which one side has a gradient mainly due to one kind of composition, the other side has a gradient due to another kind of composition, and the composition transitions from one side to the other side; 4. a structure with a small porosity layer of low porosity thick film followed by a large pore area; A structure with vertical pores with spatial organization, in which these vertical pores can act as channels for the diffusion of nutrients, 6. making these foams in a three-dimensional mold 6. a structure that forms a three-dimensional foam having a desired microstructure; It is a combination structure of composition and structural gradient.

In addition, various proteins (including short-chain peptides), proliferating agents, chemotactic agents and therapeutic agents (antibiotics, analgesics, anti-inflammatory agents, anti-rejection agents (eg immunosuppressants) and anticancer agents) Alternatively, the ceramic particles can be added to the foam during the preparation process or absorbed into or into the foam after the foam has been prepared. For example, a synthetic polymer or biopolymer (collagen, elastin, etc.) that can absorb the pores of the foam in a biologically acceptable manner
Alternatively, it can be partially or completely filled with a biocompatible ceramic material (such as hydroxyapatite), and combinations thereof (with or without materials that promote tissue growth in the device). Suitable materials include autograft, allograft or xenograft bone, bone marrow, morphogenetic protein (BMP), epidermal growth factor (EG)
F), fibroblast growth factor (FGF), platelet-derived growth factor (PDGF), insulin-derived growth factor (IGF
-I and IGF-II), transforming growth factor (TGF-β), vascular endothelial growth factor (VEGF) or other osteoinductivities known in the art.
e) or osteoconductive materials, including but not limited to. Biopolymers can also be used as delivery vehicles for conductive or chemotactic materials or growth factors. Examples include recombinant or animal-derived collagen or elastin or hyaluronic acid and the like.
A bioactive coating or surface treatment can also be applied to the surface of the material. For example, a bioactive peptide sequence (RDG) can be attached to facilitate protein absorption and subsequent attachment of cellular tissue. In addition, therapeutic agents can be provided by these foams.

In another embodiment of the present invention, the polymers and mixtures used to form the above-described foams can contain a therapeutic agent. To form such a foam, a therapeutic agent can be mixed with the above-described polymer prior to forming the foam, or the therapeutic agent can be loaded after the foam is formed. There are numerous types of different therapeutic agents that can be used in the foams of the present invention. In general, therapeutic agents that can be administered via the pharmaceutical compositions of the present invention include anti-infectives such as antibiotics and anti-virals, chemotherapeutic agents (ie, anti-cancer agents), anti-rejection drugs, analgesics and analgesic combinations. Drugs, anti-inflammatory agents, hormones such as steroids, growth factors (bone morphogenetic proteins (ie, BMP1-7), bone morphogenetic analogous proteins (ie, GFD-5, GFD-7 and GFD-8), epidermal proliferation Factor (EGF), fibroblast growth factor (i.e., FGF1-9), platelet-derived growth factor (PDGF), insulin-like growth factor (IGF-
I and IGF-II), transforming growth factors (ie, TGF-βI-III), vascular endothelial growth factor (VEGF), and other natural or genetically engineered proteins, polysaccharides, glycoproteins or lipoproteins, and the like. Including, but not limited to. These growth factors are described in Vicki Rosen and R.
Scott Thies literature ( The Cellular and Molecular Basi
s of Bone Formation and Repair , published by RG
Landes Company).

Foams containing bioactive materials can be foamed by blending one or more therapeutic agents with a polymer or solvent or polymer-solvent mixture to make the foam. . Alternatively, the therapeutic agent can be coated on the foam, preferably with a pharmaceutically acceptable carrier. Any drug carrier that does not dissolve the foam can be used. The therapeutic agent can be present as a liquid, a finely divided solid, or other suitable physical form. Generally or alternatively, the substrate will include one or more additives such as diluents, carriers, excipients, stabilizers, and the like.

[0061] The amount of therapeutic agent will depend on the particular agent used and the medical condition being treated. Generally, the amount of drug will be from about 0.001% to about 70%, preferably from about 0.001% to about 50%, most preferably from about 0.001% to about 20% by weight of the base. %. further,
The amount and type of polymer incorporated into the drug delivery matrix will depend on the desired release profile and the amount of drug used.

Upon contact with body fluids, the drug is released. When the drug is incorporated into the foam, the drug is released as the foam gradually disintegrates (primarily due to hydrolysis).
By doing so, an effective amount (eg, 0.00
A long-term (for example, 1 hour to 5000 hours, preferably 2 hours to 800 hours) drug can be supplied at a dose of 01 mg / kg / hour to 10 mg / kg / hour. Such dosage forms can be determined according to the condition of the subject being treated, the severity of the affliction, the judgment of the prescribing physician, and the like. Various formulations can be made by those skilled in the art according to the methods described above or methods analogous thereto.

The above foam may also serve as a skeleton former for artificial treatment of tissue. That is, the porous gradient structure induces cell growth. Cells are harvested from the patient (either before or during surgery to repair the tissue) and the cells are subjected to sterile processing conditions, as described in pre-existing patents (U.S. Pat. No. 5,770,417 to Vacanti). Subsequent processing can be used to obtain specific cell types (ie, pluripotent, stem and progenitor cells, such as mesenchymal stem cells described in US Pat. No. 5,486,359). Suitable cells that can be contacted or inoculated with the foam skeleton-forming material of the present invention include muscle cells, adipocytes,
Fibromyoblasts, ectodermal cells, muscle cells, osteoblasts (ie, osteocytes), chondrocytes (ie, osteochondrocytes),
Endothelial cells, fibroblasts, pancreatic cells, hepatocytes, bile duct cells, bone marrow cells, nerve cells, genitourinary cells (including nephritis cells),
And combinations thereof, but are not limited thereto.
Various cells (ie, autologous, allogeneic, xenogeneic cells, etc.) can be treated with the skeleton-forming material of the present invention. These cells may also contain inserted DNA encoding a protein capable of stimulating tissue attachment, growth or differentiation. The foam is placed in a cell culture environment and cells are seeded on or within the structure.
The foam is maintained in a sterile environment prior to implantation into a donor patient that has infiltrated the microstructure of the device with cells.
In vitro inoculation of the cells allows for faster growth and differentiation of the tissue. This cell differentiation and the ability of the tissue to form a particular extracellular matrix is important in artificially treating the tissue with a functional graft.

The user can choose to inoculate different cell types into different porous structures. Schaufer et al. Have different cell types (ie, stromal cells and chondrocytes)
Report that they can be cultured on different structures. These structures are immediately combined and a biphasic tissue structure can be generated for transplantation by returning the entire structure to the cell culture environment. Gradient structures can also be generated for co-cultured tissue scaffolds (Schaefer, D et al.). In addition, radiopaque markers can be added to the foam to perform image processing after implantation. After a predetermined in vitro development period (eg, 3 weeks), the tissue-treated graft is removed and transplanted into a patient. In the case of performing cell-free treatment, a sterilized cell-free skeleton-forming material can be used to replace damaged or damaged tissue.

The foam skeleton forming materials of the present invention may be sterilized by conventional techniques, including radiation-based sterilization (ie, gamma-ray treatment), chemical-based sterilization (ethylene oxide treatment), or other suitable treatment. Can be. This sterilization treatment is preferably performed with ethylene oxide at 52 ° C. to 55 ° C. within 8 hours. After this sterilization treatment, the foam skeleton forming material is packaged in a suitable sterilized moisture-resistant package that can be used for shipping and at a health care facility such as a hospital.

In another embodiment of the present invention, the firing pair may have a fibrous fabric fused to the top or bottom. In this way, surface properties such as porosity, permeability, disintegration rate and mechanical properties of the structure can be adjusted. The fibrous fabric can be manufactured by an electrospinning method, in which a fibrous layer can be formed on a freeze-dried foam surface. Electrospinning is a method that has been used to make fibers.
Electrospinning of many polymeric materials is described in the following patents and literature: U.S. Pat. No. 4,522,709;
Nos. 5,024,789 and 5,311,884
No. 5,522,879, “Nanometre diamet of polymer fibers produced by electrospinning.
er fibers of polymer, produced by electrospinnin
g) "(Nanotechnology, 1 (1996) pp. 216-233)," Structure and morphology of small diameter electrospun aramid fibers "
electrospun aramid fibres) "(Polymer Inter., 3
6 (1995) pp. 195-201), and "Carbon nanofibers from polyacrylonitrile and mesolayer pitch," incorporated herein by reference.
phase pitch) "(Journal of Advanced Materials, Vo
l.3, No. 1 (1999) pp. 36-41).

In this method, a power is applied to the polymer solution that overcomes the surface tension of the solution to form a charged jet. A jet of this solution is jetted onto a grounded substrate, dried and solidified. By adjusting the spinning conditions, the resulting fibers can be in the range of about 0.1 μm to about 20 μm, preferably in the range of about 0.3 μm to about 5.0 μm. In this case, it is a freeze-dried foam. Two examples of such a structure are shown in FIGS. These layer structures can be made of at least one biocompatible material and may be non-absorbable, absorbable, or a combination thereof.

The composition, thickness and porosity of the fibrous layer can be adjusted to provide the desired mechanical and biological properties. For example, the rate of bioabsorption of the fibrous layer can be selected to provide a longer or shorter bioabsorbable structure relative to the underlying foam layer. In addition, the fibrous layer may have greater integrity with the composite, so that mechanical force is applied to the fiber side of the structure. For example, sutures, staples, or other anchoring devices can be used to hold the composite in place thanks to the fibrous layers. As a general guide, but not to limit the scope of the invention in any way, it is generally preferred that the fibrous layer be comprised of a layer having a thickness of about 1 micron to 1000 microns.

One useful application of the foam structure having a fibrous surface layer is for use as a vascular graft. The layers in such a structure form a plurality of concentric cylinders,
The fibrous layer will reduce permeability. The lyophilized foam structure can be optimized for cell invasion by smooth muscle cells or fibroblasts on the adventitia surface and endothelial cells on the luminal surface. The fibrous textile layer acts as a barrier to blood leakage and enhances the mechanical properties of the structure.
In addition, the fibrous layer acts as a barrier to smooth muscle cell proliferation and prevents intimal hyperplasia.

Another useful application of such a foam / fabric hybrid structure is its use as a scaffold for breast regeneration. The fibrous layer enhances mechanical integrity,
Maintain the original shape of the skeleton forming material. When performing a biopsy or removing a tumor, the cavities can be filled with a foam scaffold that covers such fibers, into which cells or bioactive agents can be poured. You don't have to.

Another useful application of such a foam / fibrous structure is its use as a scaffold for artificial treatment of meniscal tissue. In this case, the wedge-shaped implant can be made with a fibrous covering surrounding the freeze-dried central part of the foam. Cells can be injected into the center of the implant through this fibrous layer. This textile surface layer allows for the diffusion of nutrients and waste products, but limits the migration of cells from the skeletal material.

The following examples are provided to illustrate, but not limit, the principles and implementation of the present invention. That is, many additional embodiments within the spirit and scope of the present invention are readily contemplated by those skilled in the art.

[0073]Example  In the following examples, each polymer and monomer was
Chemical composition and purity (NMR, FT-IR), thermal analysis
(DSC), molecular weight (intrinsic viscosity), and baseline and
In vitroMechanical properties (Instron stress / strain)
About.

¹ H-NMR measurements were performed at 300 MHz NMR using CDCl ₃ or HFAD (hexafluoroacetone sesqua deuterium oxide) as a solvent. In addition, thermal analysis of the separated polymer and monomer was performed by a Dupont 912 differential calorimeter (DSC) apparatus. Further, the intrinsic viscosity (IV dL / g) of each polymer and copolymer was adjusted to a concentration of 0.1 g / dL using chloroform or hexafluoroisopropanol (HFIP) as a solvent at 25 ° C. in a 50-bore immersed in a thermostatic water bath. It was measured with a Cannon-Ubbelhode dilution viscometer.

In the following examples, ε-caprolactone polymer was PCL, glycolide polymer was PGA,
(L) Certain abbreviations are used, as indicated by the lactide polymer as PLA. Further, the percentages listed above for each copolymer indicate the respective molar percentages for each component.

[0076]Example 1 Foam with irregular microstructure (unfavorable structure)
Create  Step A. 35/65 PCL in 1,4-dioxane
Preparation of a 5% w / w homogeneous solution of PGA / PGA 1 part by weight of 35/65 PCL / PGA was dissolved in 19 parts by weight
5% w / w dissolved in 1,4-dioxane
Of the polymer volume. This 35 / 65PCL / P
The GA copolymer was formed generally as described in Example 8.
Was. This solution was made in a flask with a magnetic stir bar.
Was. Mix the mixture for 60 minutes to completely dissolve the copolymer.
Gently heated to ± 5 ° C, minimum 4 hours, over 8 hours
It is preferable to stir continuously so as not to cause such a problem. afterwards,
Super coarse porous filter (Pyre with frit disk)
x viscous extraction filter paper).
Clear and uniform by filtration using dry nitrogen
A solution was obtained.

[0077]Step B. freeze drying  Freezemobile 6 from VIRTIS, a laboratory-scale freeze dryer
Was used in this experiment. Start this freeze dryer and
Keep chamber at 20 ° C under dry nitrogen for about 30 minutes
did. Install thermocouple to monitor shelf temperature
And monitored. Before actually starting the process,
Carefully mold the homogeneous polymer solution prepared in A
Filled in mold. In this experiment, a glass mold was used.
But inert to 1,4-dioxane,
It has good heat transfer properties and can easily remove foam
It can be made of any material as long as it has
Molds can be used. The gas used in this example
4. The lath mold or dish weighs 620 grams and
5mm thick optical glass with outer diameter 21cm and inner diameter
It was 19.5 cm cylindrical. Also the height of the mouth of this dish
The length was 2.5 cm. Next, the following steps are performed to
A foam having a thickness of mm was formed. (I) freezing the glass dish containing this solution at 20 ° C.
Carefully place (do not tilt) on the shelf of the sinter dryer
Was. Start the treatment process and maintain the shelf temperature at 20 ° C for 30 minutes.
To carry out thermal conditioning. (Ii) The solution is then cooled by cooling the shelf to −5 ° C.
Was cooled to -5 ° C. (Iii) After 60 minutes of freezing at −5 ° C., vacuum
To start the primary drying of dioxane by sublimation
Was. Under vacuum at -5 ° C to remove most of the solvent
One hour of primary drying is required. At the end of this drying stage
At the end of the day, the reduced pressure level is generally
(6.7 Pa) or less. (Iv) Next, reduce by 50 mTorr (6.7 Pa) or less.
Absorbed by performing secondary drying in two stages under pressure
Dioxane was removed. In the first stage, the temperature of the shelf
Was raised to 5 ° C. and maintained at this temperature for 1 hour. This first
The stage of the second drying process starts from the end of the stage. This
In the second drying step, the temperature of the shelf is set to 20 ° C.
Raised and maintained at this temperature for about 1 hour. (V) at the end of the second step, the temperature of the freeze dryer
Was returned to room temperature, and the pressure was released by supplying nitrogen. That
After that, fill the chamber with dry nitrogen for about 30 minutes
I opened the door.

The above process is suitable for producing a foam having a thickness of about 2 mm or less. In addition, if it is an expert in this technical field, the various conditions described in this specification are specific, and each operating condition is, for example, solution concentration, polymer molecular weight and composition, solution volume, molding It will be appreciated that it will vary within a certain range depending on several factors, such as mold parameters, mechanical variables such as cooling rate and heating rate. FIG. 1 shows an SEM of a cross section of a foam formed according to the method described in this example. Note the irregular microstructure (not the preferred structure) in this foam.

[0079]Example 2 Creating foam with vertical flow path  In this example, the transfer and orientation of the nutrients
Vertical flow can constitute a pathway for the regeneration of isolated tissue
Of making 35/65 PCL / PGA with road
I will tell.

The present inventor made this foam using an FTS Dura freeze dryer equipped with a computer control and data monitoring system. The first in making this foam
Is the creation of a uniform solution. That is, the first embodiment
A 10% w / w uniform solution of 35/65 PCL / PGA was prepared in the same manner as described in Step A of (a). The polymer solution was then carefully filled into the dish just before the processing step was actually started. This dish weighs 6
It was 20 grams, 5.5 mm thick optical glass, cylindrical with an outer diameter of 21 cm and an inner diameter of 19.5 cm. The height of the mouth of this dish was 2.5 cm. Next, a foam having a desired structure with a thickness of 2 mm was formed by performing the following steps. (I) The dish containing this solution was placed on the shelf of a freeze dryer precooled to -17 ° C. Starting the treatment process, the temperature of the shelf was maintained at -17 ° C for 15 minutes to quench the polymer solution. (Ii) after this 15 minute quenching to -17 ° C,
A vacuum was supplied to initiate primary drying of the dioxane by sublimation and maintained at 100 mTorr (13.3 Pa) for 1 hour. (Iii) Next, secondary drying was performed at 5 ° C. for 1 hour and at 20 ° C. for 1 hour. At each temperature, the vacuum level was maintained at 20 mTorr (2.7 Pa). (Iv) At the end of the second step, the temperature of the freeze dryer was returned to room temperature, and nitrogen was supplied to release the reduced pressure. Thereafter, the inside of the chamber was filled with dry nitrogen for about 30 minutes, and then the door was opened.

FIG. 2 is an SEM photograph showing a cross section of a foam having a vertical flow path. These channels extend through the thickness of the foam.

[0082]Example 3 Foam with structural gradient  In this example, 35 / 65ε-caprolactone-
With a 10% solution of co-glycolide,
The creation of a foam having a gradient in the form of a foam will be described. This
The method for making such a foam is with one exception
This is the same as the method described in the second embodiment. Sand
In the freeze-drying step (ii),
The time for maintaining the solution is 30 minutes.

FIG. 3 is an electron micrograph of a cross section of the foam of this example. Note the pore size and pore shape through the thickness of the foam.

[0084]Example 4 Composition gradient foam  In this example, a composition gradient
Method for making foam without morphological gradient
explain. Such foams are made of two or more polymers.
Created by polymer solution created by physical mixture
it can. This example uses 35/65 PCL / PGA and
With composition gradient created by 40 / 60PCL / PLA
The foam to be formed will be described.

Step A. 35 in 1,4-dioxane
/ 65PCL / PGA and 40 / 60PCL / PLA
Preparation of a Solution Mixture of In a preferred method, two separate solutions are used:
(A) 10% w / w polymer solution of 35/65 PCL / PGA and (b) 1 of 40/60 PCL / PLA
A 0% w / w polymer solution was first prepared. After making these solutions as described in Example 1, equal parts of each solution were poured into a mixing flask. The polymers used for preparing these solutions are described in Examples 8 and 9. A homogeneous solution of this physical mixture is gently heated to 60 ± 5 ° C. for about 2
It was obtained by continuously stirring with a magnetic stir bar for hours.

[0086]Step B. freeze drying  The inventor has a computer control and data monitoring system.
This foam is dried using an FTS Dura freeze dryer equipped with a
Created. The first step in making this foam is described above.
This is the preparation of the uniform solution described in step A. Next
Carefully note this solution just before actually starting the process.
Filled in a dish. This cylindrical glass dish weighs 1
17 grams, 2.5mm thick optical glass, outside
It has a cylindrical shape with a diameter of 100 mm and an inner diameter of 95 mm.
Was. The height of the mouth of this dish was 50 mm. Next
The following steps are performed to obtain a compositionally graded 25 mm
A thick foam was formed. (I) freezing the glass dish containing this solution at 20 ° C.
Place on the shelf of the coke dryer and melt at 20 ° C for 30 minutes.
The condition of the liquid was adjusted. Next, start the processing steps,
0.5 ° C / min cooling rate program to reduce shelf temperature
It was set to -5 ° C. (Ii) Thereafter, the solution is kept under freezing conditions (-5 ° C) for 5 hours.
I carried it. (Iii) Next, vacuum is supplied to dioxane by sublimation.
Primary drying was started and 100 mTorr (13.3P
Maintained in a) for 5 hours. (Iv) 5 hours at 5 ° C. and 10 hours at 20 ° C.
Was subjected to a secondary drying treatment. Decompression level at each temperature
It was maintained at 20 mTorr (2.7 Pa). (V) at the end of the second step, the temperature of the freeze dryer
Was returned to room temperature, and the pressure was released by supplying nitrogen. That
After that, fill the chamber with dry nitrogen for about 30 minutes
I opened the door. The resulting foam is shown in FIGS.
By examining the morphology of the foam wall as shown in
It has a gradient in the chemical composition. This chemical group
The gradient in the synthesis is based on NMR data as described below.
It was confirmed even more.

The foam sample made by the above method had a thickness of about 25 mm and was characterized in mole% composition. This foam sample is composed of a mixture of PCL / PLA and PCL / PGA. First,
A slice of the foam sample was made and analyzed to confirm that the material had a compositional gradient.
That is, these sample slices were used for (1) foam I
A (top slice), (2) foam IB (top middle slice), (3) foam IC (bottom middle slice),
And (4) Foam ID (bottom slice). Thereafter, NMR by diluting after dissolving the material of 5mg to 300μL of hexafluoro Cesc adjuvant Ute potassium oxide (HFAD) in C ₆ D ₆ of 300μL
Made a sample.

From the above NMR results, it can be seen that these foam samples have a gradient in composition.
That is, the top layer of foam has a high PLA concentration (47 mol%), while the bottom layer of the foam has a high PGA concentration (56%).
Mol%). From these results, during the freezing process, PC
It is considered that the L / PGA copolymer and the PCL / PLA copolymer formed a foam having a specific composition gradient due to the difference in their phase separation characteristics.

[0089]Example 5 Structurally graded foam  In this example, there is a compositional and structural gradient.
Of foam that does not necessarily have a morphological gradient
The method of formation will be described. There are two or more such foams
Polymer made by physical mixture of the above polymers
Can be made by solution. This example is 35/65 PCL
/ PLA (as described in Example 9) and 95
/ 5PLA / PCL (as described herein)
A random code with an IV of 1.8 in HFIP measurement
(Polymer)
Will be described. 35 / 65PCL / PLA is soft
95/5 PLA / P
CL is a relatively rigid copolymer. These two types
Compositional and structural gradients due to the combination of
Distribution can be configured. This foam is described in Example 4.
First, 1,4-di-
10% weight of 35/65 PCL / PLA in Oxane
10% weight of solution / weight solution and 95/5 PLA / PCL
Creating a uniform 50/50 physical mixture of volume / weight solutions
Started from. Foams with such a compositional gradient are bone
-Good texturing for tissue junctions such as cartilage borders
Template can be provided.

[0090]Example 6 Cell culture and differentiation data  95/5 PLA / PGA, 90/10 PGA / PLA,
95/5 PLA / PCL, 75/25 PGA / PCL
And film made by 40 / 60PCL / PLA
Was tested. Set as positive control in all assays
Woven culture polystyrene (TCPS) was used. Do the test
Prior to each polymer disc, a 24-well ultra-low
Pre-humidification for 20 minutes at the bottom of the star dish
did.

The 95 / 5PL used in this example
The A / PGA copolymer is a random copolymer having an IV of 1.76 as measured in HFIP at 25 ° C. and used in Panacryl ™ sutures (Ethicon, Somerville, NJ). Also, 90/10 PGA / PLA copolymer has H
A random copolymer having an IV of 1.74 as measured in FIP and used in Vicyl ™ sutures (Ethicon, Somerville, NJ). The 95/5 PLA / PCL polymer was made as described in Example 10 and has an IV of 2.1 as measured in HFIP at 25 ° C. Also, 75
/ 25PGA / PCL copolymer is a segmented block copolymer having an IV of 1.85 and is described in U.S. Patent No. 5,133,739. This copolymer is used in Monocryl ™ sutures (Ethicon, Inc., Somerville, NJ). further,
The 40/60 PCL / PLA copolymer is made as described in Example 9 and has an IV of 1.44.

[0092]Cell attachment and proliferation  24-well ultra low class containing each polymer
40,000 pcs / wa in a dish
Cells. This Ultra Low Cluster Day
Brush is coated with a layer of hydrogel polymer.
This coating is protein and cellular
Delays adhesion to the well. Therefore, for each biopolymer
Cell adherence was determined after 24 hours of culture (each polymer
N = 3 for-). Attached cells to trypsin treatment
More liberated and the number of cells was determined by hemacytometer.
In addition, cell growth was performed on days 3 and 6 after inoculation of cell growth.
Was evaluated.

[0093]Proliferation assay Alkaline phosphatase activity  p-Nitrophenol phosphate substrate (Sigma 104)
And alkali by colorimetric assay according to the manufacturer's instructions
Phosphatase activity was determined. That is, 4 cells
Film or mesh at a density of 0000 / well
And incubate for 1 day, 6 days, 14 days and 21 days
Was. On the sixth day, when the cells became dense, they became mineralized.
Medium (10 mM β-glycerophosphate, 50 μg
/ Ml of ascorbic acid added culture medium)
Was. Thereafter, the alkaline phosphatase activity was increased at each of the above times.
Between cells homogenate (0.05%, Triton
X-100). Pierce Micro B
Determine the amount of protein in the cell extract with the CA reagent
Was. Membrane binding with histological staining kit (Sigma)
Film and Messi against alkaline phosphatase
Primary rat osteoblasts cultured on a mouse were stained. all
For each film and mesh,
And tested three samples.

[0094]Osteocalcin ELISA  Medium by osteoblasts cultured on various films
Osteocalcin secreted into the body
Ocalcin ELISA Kit, Biomedical T, Boston
echnologies). By ELISA
Before each protein measurement,
Aliquots of the medium from the wells were lyophilized. Each po
Three samples were tested against the rimers and the ELISA
Was repeated twice.

[0095]Von Kossa staining  Three samples of von Kossa nitric acid for each polymer
The tissue mineralized by silver staining was stained.

[0096]Alkaline phosphatase and osteoca
Expression of Lucin mRNA  Extracted from cells cultured on film for 21 days
Alkaline phosphatase in cells using RNA
And osteocalcin mRNA expression are semi-quantitatively reversed
By transcriptase polymerase chain reaction (RT-PCR)
evaluated. Seven days after inoculation, the culture medium was changed to a mineralization medium.
(3 mM β-glycerophosphate and
50 μg / ml ascorbic acid was added). The cells
After culturing for 2 weeks, culturing was performed for a total of 3 weeks.
Glue using the RNeasy mini kit provided by Qiagen.
Extract total RNA from 4 samples per group
Was. Total RNA properties and properties for each polymer group
And the amount was measured. First, the reverse transcription reaction (Superscr
ipt II, Gibco) Reverse transcription of whole RNA to cDNA
Obtained. Next, osteocalcin, alkaline phosphater
Glyceraldehyde-3-phosphate-de
CDNA corresponding to hydrogenase (GAPDH) is already
Protocol (GIBCO BRL manufacturer's instruction manual)
Amplification was carried out as described in Osteocalcin, Alkaliho
Sphatases and primers for GAPDH
Sequence (Table 2) in the FASTA program (Wisconsin)
(Genetic Computer Group, Madison, NJ)
Was. Number of PCR cycles for each primer (Table 3)
Range of RNAs that are
Preliminary studies were also performed to determine the enclosure. these
PCR product is 1% (by weight) containing ethidium bromide
Electrophoresed on agarose gel. Then these games
Photographed under UV light and the male to GAPDH
Theocalcin and alkaline phosphatase mRNA
Was evaluated by densitometry.

[0097]Statistical analysis  Analysis of variance by Tukey immediate post-hoc comparison method
Significant difference for all quantitative tests using (ANOVA)
Was evaluated. Table 2 Primers used for RT-PCRGene Animal species Forward primer Reverse primer Size (bp)  Gene 1 rat 5 '5' 379 ATCGCCTATCAGCTAAT GCAAGAAGAAGCCTTT GCAC GGG gene 2 rat / 5 'CAACCCCAATTGTGA 5' 339 human CGAGC TGGTGCGATCCATCAC AGAG gene 3 mouse / 5'ACCACAGTCCATGCC 5'TCCACCACCCTGTT 452 2: Osteocalcin gene 3: GAPDH Table 3 PCR optimization cycleGene cDNA (μl) Number of cycles  Alkaline phosphatase 125 osteocalcin 135 GAPDH 123

[0098]result Cell attachment and proliferation in bioabsorbable polymers  Cell morphology between various polymers and compared to TCPS
No remarkable difference was observed. After 24 hours of culture
Adhesion to various biopolymer films in the rat
Was equivalent to TCPS. On day 3, cells were 4
All films except 0 / 60PCL / PLA
Grew well. In addition, 40 / 60PCL / PLA
In this case, the proliferation was 60% as compared with TCPS. Further
In addition, 95/5 PLA / PGA and 90/10 PGA /
PLA film is TCPS and 40 / 60PCL / P
Higher degree with significant difference (p <0.05) compared to LA
(FIG. 7).

[0099]Differentiation assay Alkaline phosphatase enzyme activity  95 / 5PLA / PGA, 90 / 10PGA / PLA
And osteoblasts cultured on 95/5 PLA / PCL film
Of alkaline phosphatase activity expressed by cells
Files are similar to profiles found in TCPS
I was similar. On the other hand, on days 14 and 21
0 / 60PCL / PLA and 75 / 25PGA / PC
In the case of osteoblasts cultured on L film,
Alkali film compared to another film and TCPS
Phatase specific activity increased with significant difference (p <0.05)
(FIG. 8).

[0100]Alkaline phosphatase and osteoca
Expression of Lucin mRNA  95/5 PLA / PGA, 40/60 PLA / PCL,
95/5 PLA / PCL film and TCPS smell
Alkaline phosphatase for cultured osteoblasts,
Osteocalcin and mRNA in GAPDH
Was evaluated by densitometry. These conclusions
The results are shown in FIG. Note that the data in FIG.
Note that the data is from a quantitative analysis. When
These data are 40/60 PCL / PLA files.
Film significantly different from TCPS (p <0.0
5) Supporting a high degree of osteocalcin expression
Is shown. On the other hand, the remaining polymer surface
Lucin and alkaline phosphatase mRNA expression
In both cases it was equivalent to TCPS.

[0101]Conclusion  Different bioabsorbable fills tested following 6 days of culture
Cell adhesion and growth between cells or mesh
No noticeable difference was seen. In addition, these results
Between each material for each differentiation property
The difference was found to be relatively clear. That is, 40
Cell culture on 14 / 60PCL / PLA film was 14
Films and TCPS after culture on days 21 and 21
Alkaline phosphatase activity improved compared to
Sex and osteocalcin mRNA expression was indicated.

For a more complete understanding of this technique,
MA Aronow, LC Gerstenfeld, TA Owen, MS
Tassinari, GS Stein, and JB Lian, "Factors that promote progr., Promote the progressive development of the osteoblast phenotype in cultured fetal rat calvarial cells.
essive development of the osteoblast phenotype in
cultured fetal rat calvaria cells) ”(Journal of
Cellular Physiology, 143: 213-221 (1990)), and Stein, GS, Lian, JB and Owen, TA, "Cell Growth to Regulate Tissue-Specific Gene Expression During Osteoblast Differentiation. Relation (Relation
ship of cell growth to the regulation of tissue-sp
ecific gene expression during osteoblast different
iation) "(FASEB, 4, pages 3111 to 3123 (1990)).

[0103]Example 7 In Vivo of Foam Mixture in Porcine Skin Wound Healing Model
Examination of  In this example, 1 in the pig full thickness resection wound model
mm and 0.5 mm thick foam skeleton forming material
The results will be described. Realize foam tissue skeleton forming material
40 / 60ε- created as described in Example 8
In caprolactone-co-lactide and in Example 9
35 / 65ε-caprolactone made as described
-Made with a mixture of co-glycolides. That is,
These polymers were mixed together with those described in Example 3.
Approximately the same (cooling rate to -5 ° C at a cooling rate of 2.5 ° C / min.
1 mm and 0.5 mm thick
A foam formed. Scanning electron micrograph of 0.5mm foam
The truth is shown in FIGS. 11A, 11B and 12. FIG. So
After, foam of two thicknesses (0.5mm and 1mm)
PDGF supply in a resected model with injured body
And these four different types tested without supply
Samples were evaluated.

48 full thickness resection wounds (4 sites per pig) in 4 pigs transplanted 8 days after wound formation
Was subjected to a random histological evaluation. This evaluation was performed on H & E stained slides. During this histological evaluation, the following parameters were ranked and / or evaluated in each sample. Qualitative and quantitative evaluation of substrate infiltration of cells; (2) infiltration of polymorphonuclear leukocytes (PMN) into the substrate contact area (ventral surface); ) Inflammation in the lower granulation tissue bed, (4) epidermal response to substrate, and (5)
This is the degree of substrate separation.

[0105]Animal management  Pigs must be caged individually (minimum floor of 10 square feet)
(Having an area) and attached an identification tag. All pigs
Were assigned individual animal numbers. That is, the animal body number
Issue, type / pedigree, date of surgery, surgical technique and duration of experiment
Tags that describe the time period and the date of euthanasia
I attached it. Each animal is identified by a permanent marker.
The body number was marked on the base of the neck.

The room for each animal is conditioned at 40% to 70% relative humidity and 15 ° C to 24 ° C (59.0 to 75.2 °).
F). Each animal was fed a standard pig meal once daily but not overnight before any experimental treatments requiring anesthesia. Water was available ad libitum. The daily light: dark cycle was 12:12 hours.

[0107]Anesthesia treatment  On the first day of the experiment, on the day of each evaluation and on the day of necropsy,
Restrain your body, Tiletamine HCL plus Zolazepam HCL (T
elazol®, Fo of Fort Dodge, Iowa
rt Dodge Animal Health, 4mg / ml) and Xyla
zine (Rompun®, Shawnee Mi, KS)
Bayer Corporation's Agriculture Department,
Animal Health, 4 mg / ml) or via the nose
Isoflurane (AErrane®, Iowa) to be administered
Fort Dodge's Fort Dodge Animal Health) Inhalation
Anesthesia was performed with any of the anesthetics (5% volume). Movement
When performing surgery on an object, the animal is passed through the nasal head.
Isoflurane (AErrane®) for inhalation anesthesia
Maintained in girder. Also, after recovering from each treatment,
Gave the thing.

[0108]Creating a surgical site  One day before the surgical procedure, the body weight was measured and the rear area of four pigs was measured.
Area with # 40 surgical shaving blade
Mowed with a ripper. Then remove the shaved skin
Shaved with a shaving cream and razor
And rinsed. This shaved skin and the whole animal
The body (excluding the head) is further washed with PCMX washing solution (Pharmase
al (R) Scrub Care (R), California
Pharmaxal Department of Baxter Healthcare, Valencia, California)
Scrub brush with a sponge
From HIBICLENS® chlorhexidine gluco
Nate (entered from COE Laboratories, Chicago, Illinois)
It was further scrubbed by hand. Sterilize this animal
Wipe dry with a towel. Then the back of each animal
Sterile NU-GAUZE gauze on top (Jo, Arlington, TX)
hnson & Johnson Medical)
RPROOF^★Tapes (Johnson & Arlington, Texas)
(Obtained from Johnson Medical). This dynamic
Apply the entire body of the object to Spandage ™ elastic bandage (new
From MediTech International in Brooklyn, York
(Obtained) and kept on a clean surface overnight.

On the day of surgery, just prior to transporting the animal to the surgical site, the skin on its back was treated with PCM, as was done the day before.
Washed again with a surgical scrub brush sponge with X Wash Solution (Pharmaseal®, Scrub Care®), rinsed and wiped dry with a sterilized towel. Place the animal on the operating table with its face down,
% Alcohol and dried with sterile gauze. Sterile surgical markers (a division of Johnson & Johnson Professional of Raynham, MA)
Labels were made on the dorsal skin according to the desired location of each total thickness wound (from Codman) and the acetate template.

[0110]Surgical treatment  After anesthesia, the left and right
12 full thickness resections in two rows parallel to the spine in the dorsal area
(1.5 cm x 1.5 cm) was created for each animal. Sa
Further, a pair of scissors and / or a scalpel is
Used for weave removal. Bleeding with a sponge tampon
Adjusted. Leave enough space between the wounds to put the wounds together
Avoided interference. Digitally measure the thickness of the resected tissue
It was measured by par.

[0111]Treatment and bandage supply  A coded treatment procedure for each wound prepared in advance (each experimental subject
Was blindfolded for all treatments)
did. Sterile individual test article (1.5 cm x 1.5 c
m for 24 hours in a sterile saline solution).
The secondary dressing was placed at the wound defect according to a predetermined plan. Next
And a non-adhesive, salt-dipped, rectangular RELEASE
^★Bandage (Johnson & Johnson, Arlington, TX)
A secondary dressing consisting of
Placed on top of the above test article. In addition, BIOCLUSIVE
^★Bandage Layer (Johnson & Joh of Arlington, TX)
nson Medical) to seal wound and wet wound
The bandage inside was maintained while keeping the air. Also, Rest
on ™ (a 3M media company in St. Paul, Minn.)
Cal-surgical part) Polyurethane self-adhesive foam
A strip is placed between each wound to allow for mutual
It prevented contamination and wound damage and bandage slippage.
In addition, NU-GAUZE^★BIOCLUSIVE layer^★Bandage and Re
Placed on top of ston (TM) foam, WATERPROOF^★The
Each bandage was secured with a band. Then the animal
Spandage (TM) elastic netting to hold each bandage
did.

The above secondary dressing was removed daily and replaced with a new RELEASE ^★ secondary dressing soaked in saline. Also, the primary bandage (test article) was not touched until the unit was replaced or pushed out of the wound defect.

[0113]Postoperative medical management and clinical observation  After surgical procedures under anesthesia, the animals are removed from each cage
And returned to recovery. Immediately after and next to each animal
On the day of painkiller (baprenorphine hydrochloride (Bu
prenex Injectable, 0.01mg / kg) UK Hull Re
(sold by ckitt & Colman). anesthesia
Signs of deep or painful behavior of each pig after recovery from
Was observed. As a result, there are no signs of pain
Did not.

After the day of surgery, each pig was observed twice a day,
Their health was determined based on general behavior, appearance, food consumption, urination and defecation, and the presence of abnormal discharge.

[0115]Euthanasia  At the end of the experiment (8 days after the injury treatment), the vein around the ear margin
Via ((Sold by Butler, Columbus, Ohio)
Socumb (TM) Pentobarbital sodium
And phenytoin sodium euthanasia solution (1 ml /
Anesthetize each animal with an intravenous injection of
Euthanized. After this euthanasia treatment, the respiratory function
Ensure that there is no pause and no palpable heart function
In order to observe the animal body. At this time, the stethoscope
Make it easier to check for outages.

[0116]Tissue sampling  Immediately after euthanasia, apply each wound to the underlying fat and small area
Was excised together with the skin. This tissue is 10% neutral buffer
In formalin chloride.

[0117]Evaluation Visual scratch assessment  Includes misalignment, wound response and physical properties of the skeletal material
General observations were recorded for days 1-3. Further
At the end of the detailed clinical evaluation, 4 to 8 days after the injury,
I went there. With or without the following parameters (Yes = 1
/ None = 0) and / or degree (with a score)
And recorded.

[0118]Bandage condition  Air exposure, test article misalignment, channeling,
Communication) and RELEASE^★Moisture content of secondary bandage
Weight (Scoring criteria: 4 = wet, 3 = wet
/ Dry 2 = dry / moist 1 =
Dry).

[0119]Wound condition  Moisture content of test article (scoring criteria: 4 = wet
3 = wet / dry 2 = dry
/ Wet, 1 = dry), inflammation (scoring group)
Criteria: 3 = severe, 2 = moderate, 1 = mild, 0 = none), re
Damage (scoring criteria: 3 = severe, 2 = moderate, 1 = light
Degree, 0 = none), coagulation, folliculitis, infection, test article height
(Scoring criteria: 4 = extremely rising, 3 = rising
, 2 = no change, 1 = down), fibri
(Scoring criteria: 3 = severe, 2 = medium, 1 = mild,
0 = none), and erythema. Also observe the color of the test article.
Was.

A excised tissue sample was taken on day 8. Whole wounds were harvested and placed in 10% neutral buffered formalin. Thereafter, the tissue was prepared in a frozen part. The tissue was excised, placed on an object holder with the Tissue-Tek® OCT compound (sold by Sakura Finetechnical, Tokyo, Japan) and quickly frozen. These samples were placed on a cryogenic bath for 10 minutes.
The cells were cut into μm and stained with a frozen H & E stain.

[0121]Histological evaluation (8 days after injury)  Histology of granulation tissue (area and length) and epidermis formation
Evaluation was performed on samples that were H & E stained at a magnification of 20 to 40 times.
Was evaluated. Dividing the height of the granulation tissue by the area divided by the length
Decided more.

Further, the histopathological evaluation of the tissue sample was performed using an H & E stained sample.
These samples were evaluated at × 2 magnification.

[0123]result  Cell immersion in the interstices of the matrix at most of all sites
Jun was seen. In most of these areas,
Junction is a changing population of fibroblasts, macrophages, and macrophages.
Composed of lophage giant cells and endothelial-like cells
True tissue ingrowth, with capillary formation
It seems like. Substrate that substantially fills the substrate in tissue
Dense fibrous connective tissue layer parenchyma on the dorsal side
0.5 mm with or without PDGF
It is found in several places in the foam. Also, 1mm
Substrate was on the surface of the tissue or was shed. Macropha
Giant cells are formed on 1mm foam skeleton forming material
More was seen in the 0.5 mm version. 1mm
If the foam is missing from the underlying granulation tissue,
In detached areas, dead infiltrating cells can
A lump of cell debris was formed.

In the case of the 0.5 mm foam scaffold, complete incorporation of the matrix into the granulation tissue bed was observed at several sites. FIGS. 13 and 14 show the incorporation of these substrates into the granulation tissue bed. FIG. 13 is a 40 × dark field micrograph of a tissue sample stained with trichrome. FIG.
FIG. 4 is a 100 × composite micrograph of a trichrome stained sample showing cell infiltration of a foam containing PDGF. Both of these photographs clearly show the complete incorporation of the substrate into the granulation tissue bed. The presence of dense fibrous tissue on the foam skeleton forming material is also clearly seen in both of these photographs. Due to their presence, 0.
5 mm foam was found to constitute a suitable matrix in epidermal tissue growth.

[0125]Example 8 Random poly (ε-caprolactone-co-glycolide)
Synthesis of  E-caprolactone-g having a molar composition of 35/65
Licolide random copolymer is synthesized by ring-opening polymerization reaction.
Done. This synthesis is described in US Pat.
No. 468,253 (herein incorporated by reference).
) Is substantially the same as the method described in (1). Add
The amount of diethylene glycol initiator
Adjust to 5 mmol / mol and dry the following properties
Got a remar. That is, the intrinsic viscosity of this copolymer
(IV) is hexafluoroisopropanol at 25 ° C.
1.59 dL / g. PCL / PGA
Is 35.5 / 64.5 by proton NMR.
And contained 0.5% residual monomer. further
The glass transition point (Tg) and melting point (T
m) at -1 ° C, 60 ° C and 126 ° C by DSC.
I found out.

[0126]Example 9 40:60 poly (ε-caprolactone-) by continuous addition
Synthesis of (co-L-lactide)  In a glove box, 100 μL (33 μmol)
0.33 mol of tin (II) octoate in toluene
Solution, 115 μL (1.2 mmol) of diethylene glycol
Cole, 24.6 grams (170 mmol) of L-lac
And 45.7 grams (400 mmol) of ε-
Caprolactone in stainless steel mechanical
Treatment with cooling and nitrogen gas blanket
And flame-dried toe neck 250 mL round bottom frus
I put it inside. The reaction flask was already set at 190 ° C.
It was kept in a fixed oil bath. Also this
In a glove box, 62.0 grams (430
(Mmol) of L-lactide by flame drying and pressure equalization
Put in the lower funnel. Wrap this funnel with heat tape
At the second neck of the reaction flask. 190 ° C
After 6 hours, the molten L-lactide is
Added into the reaction flask. The reaction was continued overnight and 19
After a total of 24 hours at 0 ° C., the reaction is left at room temperature
Cooled down. Then freeze in liquid nitrogen
The copolymer was isolated by disruption of the copolymer. The remaining gala
Debris is removed from the copolymer by a bench grinder.
Removed. The copolymer is frozen again with liquid nitrogen
I removed the paddle of the mechanical stirrer. Further
In the meantime, the copolymer was ground by Wiley Mill (mill).
Place the container in a light-weight glass wide-mouth bottle
Warmed to room temperature overnight in oven. That
Later, 103.13 grams of 40:60 poly (ε-capro
Lactone-co-L-lactide)
Vacuum at 110 ° C for 54 hours in a pan
To remove volatiles. As a result, 98.7 grams (9
5.7% by weight) was recovered after degassing.
As a result of measuring the intrinsic viscosity, CHCl_ThreeMedium (concentration
= 0.1 g / dL) was 1.1 dL / g.
FTIR (CHCl on KBr window_ThreeCass from
Film, cm^-1): 2993, 2944, 286
8, 1759, 1456, 1383, 1362, 118
4,1132,1094,870, and 756.¹H
-NMR (400 MHz, HFAD / benzene, pp
m): δ1.25, two broad lines (e);
35, 2 lines (f); 1.42, 3 lines; 1.55, 2
Main line; 2.22, 3 lines; 2.35, 4 wide lines;
4.01, 3 lines; 4.05, 3 lines; 4.2, quadruple
Lines; 5.05, 3 wide lines; 5.15, 4 lines. ¹ Polymer composition by H-NMR: 41.8% PCL,
57.5% PLA, 0.8% L-lactide, <0.1%
ε-caprolactone. DSC (20 ° C./min, first heat):
T_m= 154.8 ° C, ΔH_m= 18.3 J / g. GPC
(In THF using poly (methyl methacrylate) criteria
Molecular weight determined by :): M_w= 160,000, M_n= 1
0,000, PDI = 1.6.

[0127]Example 10 Synthesis of 95/5 PLA / PCL copolymer  In the glove box, 170 μL (1.8 mm
Diethylene glycol, 350 μL (115 μM
0.33 mol of stanna octoate toluene
Solution, 17.1 g (150 mmol) of ε-capro
Lactone, and 410.4 grams (2.85 moles)
1000m flame-dried L-lactide treated with silane
Placed in L round bottom flask. This flask is made of stainless steel
Tenless steel mechanical stirrer and dry nitrogen
Connector for decompression release to hold raw gas blanket
Data. The reaction flask was already heated to 185 ° C.
It was kept in a heated oil bath for 3 hours. That
After that, remove the flask from the oil bath and leave it at room temperature
Cool. Wrap the flask in aluminum foil and
The polymer was isolated by freezing it in liquid nitrogen.
The glass attached to the polymer was scraped off. afterwards,
This copolymer is ground in a Wiley mill,
The shaved polymer is vacuum dried at 80 ° C. for 24 hours and
Grams of copolymer were recovered. This intrinsic viscosity is chloro
2.1 d in form (25 ° C, concentration = 0.1 g / dL)
L / g. This polymer composition was analyzed by proton NMR analysis.
97.2 mol% of PLA, as determined by optical analysis, and
It was found to be 2.8 mol% PCL. Remaining goods
No mer was detected.

[0128]Example 1160 / 40PLA / PCL hybrid foam / microfiber
Synthesis of textile structure  Step A. 14% w / w of 60/40 PLA / PCL
Preparation of a homogeneous trichloroethane solution 14 parts of 60/40 PLA / PCL (IV = 1.3
4 dl / g) in 86 parts of trichloroethane solvent (TC
14% weight / weight polymer solution by dissolving in E)
A liquid was prepared. Transfer this solution to a flask equipped with a magnetic stir bar.
Prepared within. In order to completely dissolve the small polymer,
It is recommended that the mixture be stirred at 60 ° C. overnight.

[0129]Step B. Electrostatic spinning  In this experiment, an electrostatic spinner was used. Spellman high voltage
DC supply (Spellman High Voltage DC Supply) (model
Le number: RHR30PN30, Spellman high voltage
The Electronics Corporation (Spellman H
igh Voltage Electronics Corporation), New York, USA
-Highway in Hoppouge, NY, USA
It was used as a voltage source. The applied voltage and mandrel speed
Adjusted by Love View Computer Software. Spinneret
The distance to gold was adjusted mechanically.

A flat sheet of freeze-dried 60/40 PLA / PCL foam made as described in Example 1 was rolled into a cylinder. Both ends were fixed with a few drops of trichloroethane, the solvent used in spinning. The foam tube was mounted on a rotating grounded conductive mandrel. Both ends of the mandrel not covered by the foam substrate were masked with insulating tape to prevent the microfibers from being attracted to both ends. The solution was placed in the spinneret and a high voltage was applied to the polymer solution. The experiment was performed at room temperature and ambient humidity.

The processing conditions during spinning were as follows: Mandrel voltage 16,000 V Mandrel speed 100 rpm Distance between spinneret and mandrel 10 cm Temperature Room temperature Humidity Ambient humidity

A layer approximately 200 μm thick was deposited on the other side of the tube.

[0133]Example 12 Hybrid foam / woven structure encapsulated
Cultured chondrocytes show limited cell migration
You  Step A. Hybrid 60/40 PLA / PCL foam
/ Preparation of Micro-Senki Fabric Skeleton Forming Material Prepared from 60/40 PLA / PCL described in Example 1.
The freeze-dried foam was described in detail in Example 11.
As described above, 60/40 PLA / PCL in trichloroethane
Of the 60 / 40P described in Example 1 during the electrospinning of
Lyophilized foam made from LA / PCL as substrate
Used as

Step B. Hybrid 60 / 40PLA /
Seedling of chondrocytes into PCL foam / microfibril woven scaffold-forming material Chondrocytes isolated from bovine shoulder were placed in Dulbecco's modified Eagle's medium (high glucose) with 10% fetal bovine serum, 10 mM HEPES, 0.1 mM Essential amino acids, 20 μg / ml streptomycin and 0.2
Cultures were made in supplementation with 5 μg / ml amphotericin B.

[0135]Three-dimensional movement test  This assay uses a contracted collagen lattice implanted with cells.
Formation of biodegradable polymer skeleton at the center of collagen gel
Based on buried material. Skin equivalent
Lagen grid) was made from the following mixture: chondrocytes
8 ml of medium, neutralization medium (DME containing 0.1 N NaOH)
M / 10% FBS, prepared before use) 2 ml, collaboration
5 Biomedical Products (Collaborative
Rat tail type I obtained from Biomedical Products)
4 ml of collagen (3.99 mg / ml) and cartilage
2 ml of chondrocyte cell suspension suspended in cell medium. The above cells /
24-well hydrogel coated plate with collagen mixture
(Costar, Catalog # 3473)
(2 ml). Final cell concentration is 6 × 10⁶Pcs / ml
The collagen concentration was 1 mg / ml. Collage
The gel was polymerized at 37 ° C. for 1 hour. Following gel polymerization
Then, 1.5 ml of chondrocyte growth medium was applied to each well.
The medium was changed every other day. Shrunk for 7 days
Later, using a disposable biopsy punch,
A 5 mm diameter flaw was created over the entire thickness. Moist
Skeleton of approximately 5 mm in diameter and 2 mm in thickness supplied in the form
Implant the material into the center of the damaged collagen lattice
Was. Skeleton forming material from wound bed while collagen lattice contracts
50 μl to ensure that no material is extruded
Of non-cellular collagen was placed on top of each construct. this
These constructs are cultured in chondrocyte cell culture medium, and the medium is
Replaced by Shrinkage gel containing skeleton forming material
Stationary or bio in ultra low cluster dishes
Cultured in the reactor and harvested after 3 weeks.

[0136]Step C. Characterization of cells in scaffolds  Histology was applied to the cell-infiltrated scaffolding material. Skeleton
The material is fixed in 10% buffered formalin and
Embed in microfibers to create sections (hybrid foam / fine
Fibril skeleton forming material) or make frozen sections
(Control foam skeleton forming material). Sections are hematoxylin
/ Eosin (H / E, cell number and morphology), Safranin
O (SO; sulfated GAGs) and trichrome (Kola
Gen.). Cell immersion into these skeletal materials
The quantification of the swelling was determined by image analysis. Tuke in the data
A post hoc test analysis of variance was performed.

[0137]Experimental result  By quantitative evaluation of H / E sections of constructs cultured for 3 weeks
Chondrocytes infiltrated both skeleton-forming materials
Became. Hybrid cultivation in bioreactor
The cell infiltration into the foam / microfiber woven skeleton forming material is 40
/ 60PCL / PLA from infiltration into foam skeleton forming material
Was also significantly higher (P <0.05). Also, in the stationary state
In maintained cultures, hybrid foams / microfibers
Cell infiltration into the fabric skeleton forming material is 40/60 PCL /
Significantly higher than infiltration into PLA foam skeleton forming material
(P <0.05).

Cell infiltration Hybrid cells / mm² 40 / 60PCL / PLA foam / microfiber(3 weeks) Foam skeleton forming material Textile skeleton forming material  Static cell culture 50 80 Bioreactor 140 200 Cell culture

The hybrid foam / microfiber woven scaffold exhibited the highest cell density. The inventor has
Although the cell-cell interaction is presumed to be higher in the case of the hybrid foam / microfibril woven skeleton forming material than in other skeleton forming materials, this cell-cell interaction is selectively chondrocyte proliferation and hybrid. It is assumed that it may be possible to contribute to a differentiated function for the foam / microfiber fabric scaffold. It is postulated that this cell-cell interaction plays an important role in maintaining the chondrocyte phenotype. Chondrocytes are known to differentiate when they develop into a single layer of cells. When cultured at a high cell density in a monolayer, chondrocytes maintain their phenotype as indicated by the expression of type II collagen and aggrecan.

By 6 weeks, the 40/60 PCL / PLA foam scaffold had exhibited a cartilage-like morphology / properties predominantly localized within the capsule around the construct. On the other hand, in the hybrid foam / microfiber woven skeleton forming body,
The capsule was very thin and the morphology of the cells in the construct was cartilage-like and a more uniform distribution of cells was observed.

An embodiment of the present invention is as follows. (1) The bioacceptable complex according to claim 1, wherein the bioacceptable complex is bioabsorbable. (2) The bioacceptable composite according to claim 1, wherein the bioacceptable filamentous layer is bioabsorbable. (3) the biocompatible complex is an aliphatic polyester;
Poly (amino acid), copoly (ether-ester), polyalkylene oxalate, polyamide, poly (iminocarbonate), polyorthoester, polyoxaester, polyamideester, polyoxaester containing amine group, poly (acid anhydride) ), Polyphosphazene,
The biocompatible complex according to claim 1, formed from a bioabsorbable polymer selected from the group consisting of biopolymers and mixtures thereof. (4) The biocompatible complex according to the embodiment (3), wherein the bioabsorbable polymer is an aliphatic polyester. (5) The aliphatic polyester is lactide, glycolide, glycolic acid, ε-caprolactone, p-dioxanone (1,4-dioxan-2-one), trimethylene carbonate (1,3-dioxan-2-one), Alkyl derivatives of methylene carbonate, δ-valerolactone, β-butyrolactone, γ-butyrolactone, ε
-Decalactone, hydroxybutyrate, hydroxyvalerate, 1,4-dioxepan-2-one, 1,5,
8,12-tetraoxocyclotetradecane-7,14
-Dione, 1,5-dioxepan-2-one, 6,6-
The biocompatible complex according to embodiment (4), which is selected from the group consisting of homopolymers and copolymers of dimethyl-dioxepan-2-one and mixtures thereof.

(6) The biocompatible composite according to the embodiment (4), wherein the aliphatic polyester is an elastomer. (7) The elastomer is a copolymer of ε-caprolactone and glycolide, a copolymer of ε-caprolactone and (L) lactide, a copolymer of p-dioxanone (1,4-dioxan-2-one) and (L) lactide, ε-caprolactone And p-dioxanone copolymer, p-dioxanone and trimethylene carbonate copolymer,
The biocompatible complex according to embodiment (6), which is selected from the group consisting of copolymers of trimethylene carbonate and glycolide, copolymers of trimethylene carbonate and (L) lactide, and mixtures thereof. (8) The biocompatible composite according to embodiment (4), wherein a second aliphatic polyester is further present as a component of the biocompatible foam. (9) The biocompatible composite according to embodiment (4), wherein a second aliphatic polyester is further present as a component of the biocompatible filamentary layer. (10) The bioacceptability according to the embodiment (3), wherein the foam having the gradient structure of the bioacceptability transitions almost continuously in composition from the first portion to the second portion. Complex.

(11) The foam having the gradient structure of bio-acceptability is obtained from the first ratio of at least two different aliphatic polyesters in the composition from the first surface portion to the second surface portion. The biocompatible composite according to embodiment (10), wherein the transition is substantially continuous to a second ratio of different aliphatic polyesters. (12) The bioacceptability according to the embodiment (3), wherein the foam having the gradient structure of the bioacceptability transitions almost continuously in rigidity from the first portion to the second portion. Complex. (13) The bioacceptable body according to the embodiment (3), wherein the foam having the gradient structure of the bioacceptable state transitions almost continuously in the bioabsorption rate from the first portion to the second portion. Sex complex. (14) The bioacceptability according to the embodiment (3), wherein the foam having the gradient structure of the bioacceptability transitions almost continuously in flexibility from the first portion to the second portion. Complex. (15) The bioacceptability according to the embodiment (3), wherein the foam having the gradient structure of the bioacceptability transitions substantially continuously in the structure from the first portion to the second portion. Complex.

(16) From the first part to the second part, the foam having the gradient structure of the bio-acceptability is substantially continuous in structure from a substantially spherical porous shape to a vice general porous shape. (15)
2. The biocompatible complex according to item 1. (17) The biocompatible composite according to embodiment (15), wherein the size of the substantially spherical pores is about 30 μm to about 150 μm. (18) The diameter of the columnar pores is about 100 μm to about 4 μm.
00 μm and the ratio of length to diameter is at least 2
The biocompatible complex according to embodiment (15), wherein (19) The bioacceptable composite according to claim 1, wherein a therapeutic agent is further present in the foam having the gradient structure. (20) the biocompatible foam is from the group consisting of anti-infectives, hormones, analgesics, anti-inflammatory agents, growth factors, chemotherapeutic agents, anti-rejection agents, prostaglandins, RDG peptides, and mixtures thereof. 2. The sexually acceptable conjugate of claim 1, further comprising a selected agent.

(21) The growth factor is a bone morphogenetic protein, epidermal growth factor, fibroblast growth factor, platelet-derived growth factor, insulin-derived growth factor, transforming growth factor, vascular endothelial growth factor, and a mixture thereof. The biocompatible complex according to embodiment (20), wherein (22) The bioacceptable material is selected from the group consisting of a bioabsorbable synthetic polymer, a bioacceptable bioabsorbable biopolymer, a bioacceptable ceramic material, and a mixture thereof. The biocompatible complex according to claim 1, which is filled with: (23) The foam having the flow path has at least 200 μm.
The biocompatible composite according to claim 1, wherein the composite has a channel with an average length of m. (24) The bioacceptable composite according to the embodiment (23), wherein the flow channel extends substantially from the first surface portion to the second surface portion. (25) The biocompatible composite of claim 1, comprising a foam having an interconnected pore formed from a composition containing from about 30% to about 99% by weight of ε-caprolactone repeating units.

(26) The ε-caprolactone repeating unit is lactide, glycolide, glycolic acid, ε-caprolactone, p-dioxanone (1,4-dioxan-2-
On), trimethylene carbonate (1,3-dioxan-2-one), an alkyl derivative of trimethylene carbonate, δ-valerolactone, β-butyrolactone, γ
-Butyrolactone, ε-decalactone, hydroxybutyrate, hydroxyvalerate, 1,4-dioxepan-2-one, 1,5,8,12-tetraoxocyclotetradecane-7,14-dione, 1,5-dioxepane The biocompatible complex of embodiment (25), wherein the complex is copolymerized with a comonomer selected from the group consisting of -2-one, 6,6-dimethyl-dioxepan-2-one, and mixtures thereof. (27) having a first portion and a second portion, wherein the bioacceptable foam has a composition, rigidity, from the first portion to the second portion of the bioacceptable foam; The biocompatible composite of embodiment (25), wherein at least one property selected from the group consisting of flexibility, bioabsorption rate, and porous structure transitions substantially continuously. (28) The biocompatible composite according to embodiment (25), wherein the pores interconnected with each other have a pore size in a range of about 10 μm to about 200 μm. (29) The porosity of the biocompatible foam is about 20%
The biocompatible complex of embodiment (25), wherein the conjugate is in the range of about to about 98%. (30) The bioacceptable composite according to the embodiment (25), wherein the bioacceptable foam has a channel.

(31) The biocompatible complex according to the embodiment (30), wherein the average length of the flow channel is at least 200 μm. (32) The biocompatible composite according to embodiment (25), wherein the size of the substantially spherical pores is about 30 μm to about 150 μm. (33) The biocompatible composite according to embodiment (25), wherein the size of the substantially spherical pores is about 30 μm to about 400 μm, and the ratio of length to diameter is at least 2. (34) The bioacceptable composite according to the embodiment (25), wherein a therapeutic agent is further present in the foam having a gradient structure of the bioacceptability. (35) The bioacceptable composite of claim 1, wherein the bioacceptable foam is formed from an insert within the bioacceptable foam.

(36) The living body according to the embodiment (35), wherein the insert is selected from the group consisting of a film, a scrim, a woven fabric, a knitted fiber, a braided fiber, an orthopedic implant, a tube, and a mixture thereof. Complex of tolerance. (37) The bioacceptable complex according to claim 1, wherein the bioacceptable complex is formed in a three-dimensional structure. (38) The three-dimensionally shaped structure is a tubular shape, a branched tubular shape, a spherical shape, a hemispherical shape, a three-dimensional polygonal shape, an elliptical shape, a toroidal shape, a conical shape, a truncated conical shape, a pyramid shape, solid and hollow thereof. And a biocompatible complex according to embodiment (37) selected from the group consisting of: (39) The method of claim 2, wherein the biocompatible complex is bioabsorbable. (40) The biocompatible complex is an aliphatic polyester, poly (amino acid), copoly (ether-ester), polyalkylene oxalate, polyamide, poly (iminocarbonate), polyorthoester, polyoxaester, polyamide ester 3. The method of claim 2, wherein the bioabsorbable polymer is selected from the group consisting of polyoxaesters containing amine groups, poly (anhydrides), polyphosphazenes, biopolymers and mixtures thereof. .

(41) The method according to the embodiment (40), wherein the bioabsorbable polymer is an aliphatic polyester. (42) The aliphatic polyester is lactide, glycolide, glycolic acid, ε-caprolactone, p-dioxanone (1,4-dioxan-2-one), trimethylene carbonate (1,3-dioxan-2-one), Alkyl derivatives of methylene carbonate, δ-valerolactone, β-butyrolactone, γ-butyrolactone, ε
-Decalactone, hydroxybutyrate, hydroxyvalerate, 1,4-dioxepan-2-one, 1,5,
8,12-tetraoxocyclotetradecane-7,14
-Dione, 1,5-dioxepan-2-one, 6,6-
The method according to embodiment (41), wherein the method is selected from the group consisting of homopolymers and copolymers of dimethyl-dioxepan-2-one, and mixtures thereof. (43) The method according to the embodiment (42), wherein the aliphatic polyester is an elastomer. (44) The method of claim 2, wherein cells are seeded on said biocompatible complex. (45) The method according to embodiment (42), wherein cells are seeded on said biocompatible complex.

(46) The method according to claim 2, wherein the biocompatible complex is transplanted into an animal and brought into contact with cells. (47) The method according to embodiment (42), wherein the biocompatible complex is implanted in an animal and contacted with cells. (48) Seeding cells on the biocompatible complex, placing the biocompatible complex and the cells in a cell culture device, and growing the cells on the biocompatible complex The method according to claim 2. (49) Seeding cells on the bio-acceptable complex, placing the bio-acceptable complex and the cells in a cell culture device, and growing the cells on the bio-acceptable complex The method according to embodiment (42). (50) The method according to claim 2, wherein the cells are pluripotent cells, stem cells and precursor cells, and a mixture thereof.

(51) The cells are muscle cells, adipocytes, fibromyoblasts, ectodermal cells, muscle cells, osteoblasts, chondrocytes, endothelial cells, fibroblasts, pancreatic cells, hepatocytes, bile duct cells, The method according to claim 2, which is a bone marrow cell, a nerve cell, a urogenital cell, or a mixture thereof. (52) the biocompatible complex is selected from the group consisting of an anti-infective, a hormone, an analgesic, an anti-inflammatory, a growth factor, a chemotherapeutic, an anti-rejection, a prostaglandin, an RDG peptide, and a mixture thereof. 3. The method of claim 2, comprising a selected agent. (53) The method of claim 2, wherein the biocompatible complex is formed by a composition containing about 30% to about 99% by weight of ε-caprolactone repeating units. (54) The ε-caprolactone repeating unit is lactide,
Glycolide, glycolic acid, ε-caprolactone, p-
Dioxanone (1,4-dioxan-2-one), trimethylene carbonate (1,3-dioxan-2-one), an alkyl derivative of trimethylene carbonate, δ
Valerolactone, β-butyrolactone, γ-butyrolactone, ε-decalactone, hydroxybutyrate, hydroxyvalerate, 1,4-dioxepan-2-one, 1,5,8,12-tetraoxocyclotetradecane-7, Embodiment (53) wherein the copolymerization with a comonomer selected from the group consisting of 14-dione, 1,5-dioxepan-2-one, 6,6-dimethyl-dioxepan-2-one, and mixtures thereof. Biocompatible complex. (55) The method according to embodiment (53), wherein cells are seeded on said biocompatible complex.

(56) The method according to the embodiment (53), wherein the biocompatible complex is transplanted into an animal and brought into contact with cells. (57) Seeding cells on the biocompatible complex, placing the biocompatible complex and the cells in a cell culture device, and growing the cells on the biocompatible complex The method according to embodiment (53). (58) The method according to embodiment (57), wherein said cells are pluripotent cells, stem cells and precursor cells, and mixtures thereof. (59) the cells are muscle cells, fat cells, fibromyoblasts,
Embodiment (57) which is an ectodermal cell, a muscle cell, an osteoblast, a chondrocyte, an endothelial cell, a fibroblast, a pancreatic cell, a hepatocyte, a bile duct cell, a bone marrow cell, a nerve cell, a genitourinary cell and a mixture thereof. The method described in.

[0153]

Thus, according to the present invention, there is provided a bio-acceptable and bio-absorbable foam having a continuous gradient structure of form, structure and / or material composition in which tissue repair or regeneration can be effectively performed. it can.

[Brief description of the drawings]

FIG. 1 is a scanning electron micrograph of a cross section of an irregular microstructured foam made with a 5% solution of 35 / 65ε-caprolactone-co-glycolide copolymer.

FIG. 2 is a scanning electron micrograph of a cross section of a foam having a vertically open channel made of a 10% solution of 35 / 65ε-caprolactone-co-glycolide copolymer.

FIG. 3 is a scanning electron micrograph of a cross section of a structurally gradient foam made with a 10% solution of 35 / 65ε-caprolactone-co-glycolide copolymer.

FIG. 4 is a scanning electron micrograph of a cross-section of a foam having a gradient structure made with a 50/50 mixture of 40 / 60ε-caprolactone-co- (L) lactide copolymer and 35 / 65ε-caprolactone-co-glycolide copolymer. is there.

FIG. 5 is a scanning electron microscope of the top cross section of a foam having a gradient structure made with a 50/50 mixture of 40 / 60ε-caprolactone-co- (L) lactide copolymer and 35 / 65ε-caprolactone-co-glycolide copolymer. It is a photograph.

FIG. 6 is a scanning electron microscope of the bottom cross section of a foam having a gradient structure made with a 50/50 mixture of 40 / 60ε-caprolactone-co- (L) lactide copolymer and 35 / 65ε-caprolactone-co-glycolide copolymer. It is a photograph.

FIG. 7 is a graph of various data in cell culture.

FIG. 8 is a graph of various data in cell culture.

FIG. 9 is a graph of various data in cell culture.

FIG. 10 is an anatomical diagram of cartilage tissue.

FIG. 11: Made with a 50/50 mixture of 35 / 65ε-caprolactone-co-glycolide copolymer and 40 / 60ε-caprolactone-co- (L) lactide copolymer having a structure suitable for use as a skin skeleton forming material. 5 is a scanning electron micrograph of a 0.5 mm foam obtained. FIG. 11 (A) is a scanning electron micrograph showing the porosity of the surface of the skeleton forming material preferably facing the wound bed, and FIG. 11 (B) is preferably the skeleton forming facing away from the wound bed. 5 is a scanning electron micrograph showing the porosity of the surface of the material.

FIG. 12: Made with a 50/50 mixture of 35 / 65ε-caprolactone-co-glycolide copolymer and 40 / 60ε-caprolactone-co- (L) lactide copolymer having a structure suitable for use as a skin skeleton forming material. 5 is a scanning electron micrograph of a 0.5 mm foam obtained. It is a scanning electron micrograph which shows the cross section of the skeleton forming material which has the flow path extended in the thickness direction of a foam.

FIG. 13 is a 40 × dark field micrograph of a trichrome stained sample showing cell infiltration of the foam shown in FIGS. 11 (A) to 12 on day 8 after transplantation into a pig model.

FIG. 14. Further PDGF on day 8 after transplantation in a pig model
FIG. 13 is a composite micrograph (× 100) of a trichrome-stained sample showing cell infiltration of the foam shown in FIGS.

FIG. 15 is a photomicrograph of a cross section of an electrospun fiber layer fused to a foam substrate (both materials are 60/40 copolymers of (L) lactide and ε-caprolactone).

16 is a micrograph of the surface porous structure of the electrospinning layer shown in FIG.

 ──────────────────────────────────────────────────続 き Continued on the front page (72) Inventor Marty-N-Bacarnum, United States, 10025 New York, New York, Apartment 42, West One Hundred and Elevens Street 529 (72) Inventor Mark Sea Zimmerman, United States, 08816 New Jersey, East Brunswick, Eight Gate Road 21 (72) Inventor Angelo George Scoperyanos United States, 08889 New Jersey, White House Station, John Stevens Road 7 (72) Inventor Mora She Merican Bridgewater, 08807 New Jersey, United States -Johnson Circle 2701 (72) Inventor Crairen A. Basiglio, Deborah Court, Plainfield, 07062 NJ, USA 82 (72) Inventor Mark Bee Roller, United States, 08902 NJ, North Brunswick, Queens Place 9 (72) Inventor David Buoy Gorky United States, 08822 Copper Penny Road, Flemington, New Jersey 18 (72) Inventor Ixor Chun United States, 08822 New Jersey , Flemington, Spruce Coat 253 F term (reference) 4C081 AB04 AB11 AB19 CA171 CB011 DA01 DA02 DB01 DB04 DC04

Claims (4)

[Claims] 1. A foam and flow comprising a first biocompatible filamentary layer and a second biocompatible foam layer attached thereto, said biocompatible foam having a gradient structure. A biocompatible composite selected from the group consisting of a foam having a tract, wherein the foam having a gradient structure has a first portion and a second portion, and wherein the first portion and the second Over at least one portion, at least one property selected from the group consisting of composition, stiffness, flexibility, bioabsorption rate, and porous structure transitions almost continuously,
In addition, the foam provided with the flow path is a bio-acceptable composite having a first surface and a second surface provided with a flow path. 2. A method of repairing or regenerating a tissue, comprising:
Contacting the cell with a biocompatible complex,
The biocompatible composite comprises a first biocompatible filamentary layer and a second biocompatible foam layer attached thereto, wherein the biocompatible foam comprises a gradient structure. A biocompatible composite selected from the group consisting of a body and a foam having a flow path, wherein the foam having the gradient structure has a first portion and a second portion, wherein the first portion From the second part to the composition, rigidity,
At least one property selected from the group consisting of flexibility, bioabsorption rate, and porous structure transitions almost continuously, and the foam including the flow path has a first surface portion and a flow path. And a second surface portion. 3. A biocompatible foam having a first surface and a second surface, wherein said first surface and said second surface are formed.
A biocompatible foam having pores and channels communicating internally between the surfaces of the foam. 4. A biocompatible foam comprising a foam having interconnected pores formed by a composition containing from about 30% to about 99% by weight of ε-caprolactone repeating units.