WO1999033447A2

WO1999033447A2 - Tissue regeneration at internal wound sites

Info

Publication number: WO1999033447A2
Application number: PCT/US1998/027596
Authority: WO
Inventors: Ioannis V. Yannas
Original assignee: Massachusetts Institute Of Technology
Priority date: 1997-12-30
Filing date: 1998-12-24
Publication date: 1999-07-08
Also published as: AU2095199A; WO1999033447A3

Abstract

Disclosed is a method of promoting tissue regeneration at a wound site on an internal organ in a subject. The method comprises applying a porous biodegradable implant to said wound site after injury. The implant has the following characteristics: a) the pores of said implant have an average diameter of between about 1 νm and about 300 νm; b) the implant has a minimum water content of at least about 80 %; and a minimum specific surface area of at least about 10?3 mm2 per cm3¿; c) between about 20 % to about 80 % by weight of the implant is biodegraded at the wound site during the time period required for a wound of about the same severity, size and tissue type to complete about one half of the contraction which normally takes place in the absence of the implant; d) the implant comprises: i) a three-dimensional network of polymers which is substantially insoluble under physiological conditions; and ii) one or more specific cell-binding fragments. Also disclosed is a porous biodegradable implant having the characteristics described above, wherein the implant is a microparticle having an average diameter from about 10 νm to about 1000 νm. Also disclosed is a method of promoting tissue regeneration at an internal wound site in a subject. The method comprises applying the porous biodegradable microparticles to the wound site after injury.

Description

TISSUE REGENERATION AT INTERNAL WOUND SITES

BACKGROUND OF THE INVENTION

The adult mammal generally does not regenerate internal organs which may have lost mass due to disease or trauma. Loss of internal organ mass due to trauma (accidental or surgical) is typically accompanied by contraction of the lesion and synthesis of scar tissue. Methods of treating internal injuries which promote tissue regeneration and inhibit wound contraction and scar formation would greatly improve the clinical outcomes of patients with these injuries.

Although tissue regeneration methods have been reported for a limited number of internal tissue types, for example, nerves (U.S. Patent No. 4,955,893), the knee meniscus (Stone et al., Arthroscopy 5: 152 (1989) and Stone et al, Clin. Orthop.

252:129 (1990)) and periodontal tissue and blood vessels (U.S. Patent 4,947,849), there is still a need for tissue regeneration methods at other internal wound sites.

There is also a need for improved methods of inducing tissue regeneration at internal wound sites in which the additional trauma associated with traditional surgery is reduced or eliminated.

SUMMARY OF THE INVENTION

Disclosed herein are methods of promoting tissue regeneration and reducing scar formation by preventing contractile cells in the vicinity of a wound site (accidental or surgically induced) on an internal organ from inducing contraction at the lesion site. Also disclosed are certain biodegradable microparticles, which are able to coat internal wound sites and to bind contractile cells in the vicinity of the wound site, thereby inhibiting wound contraction and promoting tissue regeneration. One embodiment of the present invention is a method of promoting tissue regeneration at a wound site on an internal organ in a subject. The method comprises applying a porous biodegradable implant to the wound site after injury. The implant binds cells which induce wound contraction and has the following characteristics: a) the pores of said implant have an average diameter of between about 1 μm and about 300 μm; b) the implant has a minimum water content of at least about 80 % ; and a minimum specific surface area of at least about 10³ mm² per cm³; c) between about 20% to about 80% by weight of the implant is biodegraded at the wound site during the time period required for a wound of about the same severity, size and tissue type to complete about one half of the contraction which normally takes place in the absence of the implant; d) the implant comprises: i) a three dimensional network of polymers which is substantially insoluble under physiological conditions; and ii) one or more specific cell-binding fragments. Another embodiment of the present invention is a porous biodegradable implant having the characteristics described above, wherein the implant is a microparticle having an average diameter from about 10 μm to about 1000 μ . Another embodiment of the present invention is a method of promoting tissue regeneration at an internal wound site in a subject. The method comprises applying the porous biodegradable microparticles described above to the wound site after injury.

The methods disclosed herein can be used to promote organ regeneration at internal wound sites while minimizing wound contraction and scar tissue formation. When the biodegradable microparticles are used, the disclosed methods have the advantage that the microparticles can be delivered to an internal wound site endoscopically or laparoscopically, thereby minimizing the trauma of surgery. DETAILED DESCRIPTION OF THE INVENTION

The invention is a method of inducing tissue regeneration at a wound site on an internal organ with implanted structural templates. As used herein, a "wound site on an internal organ" refers to injured internal tissue other than skin, periodontal tissue, nerve tissue, the knee meniscus and blood vessels, and excludes external organs such as the skin. Examples include, but are not limited to, internal organs such as the organs of the gastrodigestive tract (e.g., esophagus, stomach, duodenum, small intestines, colon and recto-anal junction), the urinary system (kidney, ureter, bladder and urethra), the reproductive system (testes, ductuli efferentes, epididymis, ductus deferens, ejaculatory duct, seminal vesicles, prostrate gland, penis, glans of cowper, ovary, oviduct, uterus and vagina), the immune system (spleen, thymus and lymph nodes), the respiratory system (nasal cavity, trachea, bronchi, bronchioli, alveoli and visceral pleura), the endocrine glands (pituitary, thyroid, parathyroid, adrenal, endocrine pancreas and pineal), the heart, heart valves, lymph vessels, liver, gall bladder, exo-pancreas, spleen, bone, tendon, muscle, intervertebral joints, salivary glands, olfactory receptors, eyes and ears. Preferably, the structural templates, also referred to as "implants" or "regeneration templates", are microparticles having an average diameter of from about 10 μm to about 1000 μm, preferably from about 30 μm to about 500 μm. Because of their small size, microparticles can be conveniently delivered to a wide variety of wounds in a subject to promote tissue regeneration at internal wound sites, including at internal wound sites such as the internal organs described above, the knee meniscus, periodontal tissue, nerves and blood vessels, and at external wound sites such as the skin. The templates can promote regeneration at a wide variety of different internal tissue, provided that the templates have certain structural features which are described below in detail. Some adjustment of these parameters may be required for optimal performance at different tissue types.

Regeneration templates require a three-dimensional network with chains (or a scaffold comprising polymers) which is substantially insoluble in physiological solution. The three-dimensional network incorporates a high density of molecular fragments with high-affinity for cell binding. To provide for a high density of cell binding regions, the surface area of regeneration templates is preferably maximized. Large surface areas are provided by "open" network structures, for example, by porous network structures, which allow for the migration of cells inside the network. When placed at an internal wound site, contractile cells in the vicinity of the wound bind to the three-dimensional network and are thereby prevented from forming the highly specialized cell clusters which initiate wound contraction. It has been discovered that inhibiting the formation of these cell clusters leads to tissue regeneration at these anatomical sites rather than wound contraction and scar tissue formation. Preferably, binding is continued for a duration corresponding to the period of time required to complete about one half of the contraction in an untreated wound of about the same severity, size and tissue type as the wound being treated. Much longer durations interfere with the formation of new tissue at the wound site and also can result in the formation of scar capsules. Thus, regeneration templates preferably biodegrade at a rate such that contractile cells are sequestered for a sufficient period of time to promote tissue regeneration, but not so long as to impede wound healing.

As discussed above, regeneration templates are preferably porous. The pore diameter should be large enough so that cells can migrate into the interior of the network, but not so large that the specific surface area is decreased. Typically, the average pore diameter is between about 1 μm and about 300 μm, preferably, between about 10 μm and about 150 μm.

The average pore diameter can be determined by methods disclosed in U.S. Patent No. 4,947,840, the entire teachings of which are incorporated herein by reference. A sample of the regeneration template is embedded in methacrylate, sectioned to a suitable thickness (e.g. , 5 μm), mounted on a glass slide and stained with 1 % toluidine blue. The slides are then examined under a light microscope at a suitable magnification (e.g., about 125x) and field of vision (e.g., a diameter of about 0.138 inch). The number of pores across the field are counted at various locations on the slide and at different orientations. Pore size is calculated at the field size by the number of pores across the field. The degree of porosity is also defined by the water content and specific surface area. The water content, also referred to as "the pore volume fraction", is the percentage of the total volume of the material occupied by pore space and is defined by the following equation:

water content (or pore volume fraction) = V/V_tota, %

V_lotal is the total volume of the microparticle swollen in water and includes the volume of the solid as well as the volume of the water contained in the solid; V is the volume of water contained in the solid. The "specific surface area" includes the area of the external surfaces of the regeneration template as well as the internal surfaces, for example, the surfaces of the pores. The water content and specific surface area should be above a minimum to allow a sufficient density of cell binding fragments. Typically, regeneration templates have a water content or pore volume fraction greater than about 80%, preferably above about 95%, and a specific surface area greater than about 10³ mm² per cm³. The pore volume faction is determined by measuring the area fraction occupied by the pores. The pore volume fraction equals the area faction occupied by the pores, provided that the pore distribution around the sample is random, as described in Fischmeister, "Proceedings Int. Symp. RILEM/I-UPAC," Prague, Sept, 18-21, 1973, Final Report Part II, p. C-439, the entire teachings of which are incorporated herein by reference. The pore area fraction can be readily determined from the average pore diameter, described above.

The specific surface area can be determined from the average pore diameter by assuming that the pores are cylindrical, as described in Chang, Albert S.-P., Masters of Science Thesis, Department of Mechanical Engineering, Massachusetts Institute of Technology, May, 1988. The specific surface area can be also be determined by the Brunauer-Emmet-Teller method described in Chapter 11 of Moore, "Physical Chemistry, Fourth Edition", Prentice-Hall (1972). The entire teachings of Chang and Chapter 11 of Moore are incorporated herein by reference. A regeneration template comprises a three-dimensional network of polymers which is substantially insoluble under physiological conditions. "Substantially insoluble" means that greater than about 90% , preferably greater than about 98 % , of the mass of the regeneration template is retained, i.e., does not dissolve or degrade, over a period of about one to two days when the template is suspended in physiological solution.

Suitable polymers include collagen, glycosaminoglycan, proteoglycans, fibronectin, laminin, merosine, chondronectin or combinations thereof. The three dimensional network of a regeneration template can also include combinations of the polymers listed above. Preferably, the polymers are crosslinked. The degree of crosslinking influences the biodegradation rate, as discussed in greater detail below.

In a preferred embodiment the three dimensional network is collagen based, i.e., contains at least about 40% collagen. More preferably, the three dimensional network is crosslinked collagen and or a crosslinked collagen based network covalently bonded with glycosaminoglycan (GAG). Typically, the average molecular weight between crosslinks in collagen or collagen based networks is about 10 kilodaltons (kD_a) to about 40 kD_a and the amount of bonded GAG is between about 0.1 to about 20 weight percent.

Covalent crosslinking can be achieved by many specific techniques with the general categories being chemical, radiation and dehydrothermal methods. One suitable method for covalently crosslinking the polymers such as collagen-GAG composites is known as aldehyde crosslinking. In this process, the materials are contacted with aqueous solutions of aldehyde, which serve to crosslink the materials. Suitable materials include formaldehyde, glutaraldehyde and glyoxal. Glutaraldehyde is preferred because it yields the desired level of crosslink density more rapidly than other aldehydes and is also capable of increasing the crosslink density to a relatively high level. It has been noted that immersing the composites in aldehyde solutions causes partial removal of the polysaccharide component by dissolution thereby lessening the amount of polysaccharide in the final product. Unreacted aldehydes should be removed from the crosslinked material by exhaustive rinsing with water since residual aldehydes are quite toxic. Other suitable chemical crosslinking techniques include carbodiimide coupling, azide coupling and diisocyanate crosslinking.

Another crosslinking method is referred to herein as a dehydrothermal process. In dehydrothermal crosslinking, no external crosslinking agents need to be added to the composite. Rather, the composite is dehydrated to a moisture content of less than about 1 % . The actual amount of water which must be removed will vary with many factors, but, in general, sufficient water to achieve the desired crosslinking density must be removed. Thus, a collagen-GAG product, for example, can be subjected to elevated temperatures and/or vacuum conditions until the moisture content is reduced to extremely low levels and the desired crosslinking density is achieved. In the absence of a vacuum, temperatures above about 80° C, and preferably above about 90° C can be used. On the other hand, if the process is to be performed at approximately 23° C, a vacuum of at least 10^"5 mm Hg, and preferably below about 10^"6 mm Hg is suitable. In the preferred embodiment of this process, elevated temperatures and vacuum can be used in combination to expedite crosslinking. With a vacuum of at least about 10^"5 mm Hg, it is preferred to use a temperature of at least about 35° C. In general, the materials are subjected to the elevated temperatures and vacuum conditions until the desired degree of crosslinking density is achieved. The higher the temperature, the lower is the vacuum required to arrive at a given crosslink density; and vice versa. This dehydrothermal crosslinking process overcomes certain disadvantages of the aldehyde crosslinking method and produces composites having relatively large amounts of GAG strongly bound to the collagen chain. For a more detailed description of dehydrothermal crosslinking, see Yannas, I.V. and Tobolsky, A.V. , "Crosslinking of Gelatin by Dehydration,"

Nature 215(5100) :509-510, July 29, 1967, the teachings of which are hereby incorporated by reference.

Other suitable polymers include a polymer or co-polymer comprising monomer units selected from an α-hydroxy acid, a cyclic dimer of an α-hydroxy acid or a lactone. Examples of suitable α-hydroxy acids include gly colic acid and lactic acid; examples of suitable cyclic dimer s include cyclic dimer s of lactic and gly colic acid; and e-caprolactone is an example of a suitable lactone. The polymer can optionally include plasticisers.

The three dimensional network of the regeneration template also includes specific cell binding fragments which allow contractile cells to bind with the template so that the contractile cells are not able to initiate wound contraction. Examples of specific cell binding fractions include: 1) the fibronectin binding region of collagen or oligopeptides present in the fibronectin binding region of collagen which contain the sequence GTPGPQGIAGQRGVV (SEQ ID NO. : 1); 2) cell binding regions of fibronectin or oligopeptides which include the sequence RGDS; or 3) proteoglycans and glycosaminoglycans with high degrees of sulfation (e.g., chondroitin 6-sulfate, chondrotin 4-sulfate, heparan, heparan sulfate, keratan sulfate, dermatan sulfate, chitin or chitosan). With respect to polymers and copolymers comprising monomer units selected from α-hydroxy acids, cyclic dimers of α-hydroxy acids or lactones, the cell binding fraction can be connected to the hydroxyl or carboxylic acid group at either termini by means of an ester or amide bond, respectively.

The regeneration templates of the present invention have a defined biodegradation rate at the wound site. Specifically, between about 20% to about 80% by weight, preferably between about 40% and about 60% by weight, of the regeneration template is biodegraded during the time period required for a wound of about the same severity, size and tissue type to complete about one half of the contraction which normally takes place in the absence of the template. It is to be understood that the contraction rate can depend on the anatomical site of the wound and the wound severity. Thus, adjustment of the biodegradation rate of the template according to the particular tissue and wound severity may be necessary.

The contraction rate of a wound of a given severity and tissue type can be readily determined by observing the rate of contraction of similar wounds in other subjects. Preferably, the contraction rate of wounds of varying severity and tissue type can be tabulated for future use in selecting regeneration templates of the appropriate biodegradation rate. The degree of crosslinking, i.e., the crosslink density, is an important parameter in controlling the degradation rate of the regeneration template. Generally, the greater the crosslink, the lower the degradation rate, and vice versa. The molecular weight between crosslinks can be determined by the M_c test, in which strips of the material being tested are gelatinized in 80° C normal saline and the equilibrium tensile stress is studied as a function of equilibrium strain. The gelatininized material is modeled as a swollen, ideal rubber. The M_c can be calculated from the equilibrium stress-strain relation of the material. Molecular weight between crosslinks is an inverse measure of crosslink density. A more detailed discussion of the M_ctest is found in U.S. Patent No. 4,060,081, the entire of which are incorporated herein by reference.

The degradation rate is also controlled by the quantity of blocking groups which are bonded to sites on the polymers such that the ability of degradation enzymes to cleave or otherwise digest the polymer is inhibited. The degradation rate is decreased as the quantity of bonded blocking groups increases, and vice versa. Polymers blocked with groups such as GAGs can be prepared by reacting the GAG with the polymer under acidic conditions, as described in U.S. Patent No. 4,458,678 to Yannas and Burke, the entire teachings of which are incorporated herein by reference. Specific conditions for "blocking" collagen with chondroitin 6-sulfate are described in Example 1.

In the case of collagen or collagen based networks, the preferred blocking group is a GAG. Generally, collagen/GAG networks having an average molecular weight of about 10 kD_a to about 40 kD_a and an amount of bonded GAG between about 0.1 to about 20 weight percent have biodegradation rates suitable for use in regeneration templates for internal tissues.

With respect to polymers and co-polymers comprising monomer units selected from α-hydroxy acids, cyclic dimers of α-hydroxy acids or lactones, the degradation rate varies according to the proportion and type of monomer units present in the polymer. For example, in lactic/gly colic acid co-polymers, the degradation rate increases as the amount of glycolic acid in the co-polymer increases, and vice versa. Any in vitro assay which correlates to the in vivo biodegradation rate can be used to select three dimensional networks which are suitable for promoting tissue regeneration at a given internal wound. For example, the in vivo biodegradation rate of collagen and collagen based networks is known to correlate with an in vitro collagen degradation assay, which is described in Example 2, U.S. Patent No. 4,947,840 to Yannas et al., and Yannas et al., J. Biomed. Mater. Res. 9: 623 (1975). The entire teachings of these two references are incorporated herein by reference. This assay can be used to select collagen and collagen based regeneration templates having a suitable biodegradation rate for use at a given internal wound site. The units measured using this assay are referred to as

"enzyme units" . Enzyme units are a measure of the in vitro degradation rate of the material being tested. The higher number of enzyme units, the greater the degradation rate. A degradation rate below about 140 enzyme units, preferably below about 120 units is suitable for use in most internal wounds. In another example, the degradation rate of lactic/gly colic acid co-polymers in physiological solution correlates with the in vivo degradation rate. The rate of degradation can be determined by any suitable means, including by suspending a sample in aqueous medium and, after a period of time, centrifuging and determining the remaining mass of the sample by determining through spectroscopic or chromatographic means the amount of degraded product in the supernatant. In another example, the degradation rate of a three dimensional network in an aqueous medium with a pH of, for example, from about 2-3 is determined and correlated to the in vivo biodegradation rate in a suitable animal model. In the method of the present invention, the regeneration template is applied to the internal wound site. A regeneration template can be applied to a wound site in any suitable manner, including by directly contacting the wound with the template. For example, the regeneration template can be used to "coat the wound", which refers to applying the regeneration template in such a manner that the wound is at least partially covered by the regeneration template. Preferably, the wound site is substantially or completely covered with the regeneration template. Alternatively, the wound can be contacted with a regeneration template in the form of, for example, a prosthesis, a scaffold which mimics the shape of the damaged tissue or a plug in a cylindrical tube. Regeneration templates in the form of a plug can be used to promote regeneration of severed tissue, for example, a severed tendon, by placing the plug between the severed tendon and contacting each end of the plug with an end of the severed tissue. "Applying the template to a wound site" also refers to placing the template in close enough proximity to the wound so that the contractile cells which normally promote wound contraction bind to the template and are thereby inhibited from promoting contraction of the wound.

The regeneration template can be in any suitable form, for example, a membrane, sheet, tube or cylinder. Procedures for preparing a regeneration template in the form of a membrane are described in Example 1. Alternatively, the regeneration template can be one or more microparticles having an average diameter of between about 10 μm to about 1000 μm, preferably from about 30 μm to about 500 μm. Microparticles can be prepared, for example, from macroscopic regeneration templates by any suitable technique, for example, by cutting, grinding, milling or pulverizing. The shape of the microparticle is not critical and can be irregular or regular (e.g., spherical). Preferably, the microparticles are suspended in a physiologically acceptable solution such as sterile water, saline, dimethyl sulfoxide, glycerol, Ringer's solution, or isotonic sodium chloride solutions.

The regeneration template can be applied to the internal wound site using standard surgical procedures, for example, by a surgical incision suitable for exposing the injured site. The regeneration template can then be applied to the wound site manually and optionally sutured or stapled to the tissue. A suspension of microparticles can be applied to the wound site, for example, by painting, spraying, spreading, pipetting or syringing. Preferably, a tube or system of tubes which have reached the injured site are used as conduits for delivery of a suspension of microparticles by minimally invasive surgery, for example, endoscopy or laparoscopy. The regeneration template should be applied to the internal wound site as soon after injury as possible, and preferably within about five days. If the application of the regeneration template is delayed too long, it may be necessary to surgically remove tissue which has already formed. Once the tissue has been removed, the regeneration template can be applied, as described above.

A "subject" refers to a human or animal. An "animal" refers to veterinary animals, such as dogs, cats, horses, and the like, and farm animals, such as cows, pigs, guinea pigs and the like.

The invention is illustrated by the following examples which are not intended to be limiting in any way.

EXEMPLIFICATION

Example 1 - Preparation of Collagen/GAG Network Bovine hide collagen, chondroitin-6-sulfate (C-6-S, 0.11 % w/v), acetic acid (0.05M, pH 3), deionized water, isopropanol (70%), and phosphate-buffered saline were used in the manufacture and processing of the collagen-GAG membranes. The crosslinking agent was glutaraldehyde (reagent grade, Aldrich Chemical Co.), Milwaukee, WI) diluted in 0.05M acetic acid.

1. Blend 1.65 g of milled collagen with 600 ml of 0.056 M acetic acid (pH 3) for 1 hour at 4° C. 2 Dropwise add 120 ml of C-6-S solution to the blending collagen dispersion over 15 minutes at 4° C.

3. Blend an additional 15 minutes at 4° C.

4. Centrifuge at 1500 g at 4° C for 105 minutes.

5. Decant 420 ml of supernatant. 6. Reblend slurry for 15 minutes at 4° C.

7. Pour into stainless-steel trays (2 ml per square inch of tray surface); freeze 1 hour at -40° C.

8. Allow to lyophilize (freeze-dry) 24 hours at 0° C and 100 mtorr. 9. Place foam in vacuum oven at 105° C, 50 mtorr for 24 hours (dehydrothermal treatment).

10. Seal and store in desiccator until ready for next step.

11. Rehydrate in 0.05 M acetic acid (pH 3) for 24 hours. 12. Crosslink in 0.25% glutaraldehyde (pH 3) for 24 hours.

13. Rinse thoroughly with deionized water.

14. Immerse in deionized water for 24 hours.

15. Store in 70% isopropanol at 4° C.

Steps 10 through 15 are generally done with sterile technique. Membranes containing different amounts of GAG were prepared by using different concentrations of C-6-S for Step 2 discussed above. For membranes containing no GAG (i.e. , crosslinked collagen) Step 4 was changed to centrifugation at 17,000g at 4° C for 2 hours. This was necessary due to the reduction in density of the collagen dispersion caused by the absence of GAG. One method of varying crosslink density was to vary the length of crosslinking time in 0.25% glutaraldehyde (Step 12). Another way was to multiply the concentration of glutaraldehyde ten times to 2.5% while keeping the crosslinking time at 24 hours.

A third, completely different method of inducing very high crosslink densities was crosslinking with glutaraldehyde vapor at controlled temperature and relative humidity.

Example 2 - In Vitro Collagen Degradation Assay

The biodegradation rate of the various regeneration template materials was characterized using a modified collagenase assay from the Sigma Chemical Company (1977). In preparation for the assay, the materials were first refreeze- dried (Steps 7 and 8 in Example 1).

In the assay, the material to be tested was incubated for 5 hours with collagenase from Clostridium bistolyticum (GIBCO, Grand Island, NY). This bacterial collagenase hydrolyzes proteins containing proline. The amino groups liberated were measured as equivalents of L-leucine using a colorimetric ninhydrin method. The darker the final solution, the higher the concentration of free amino groups, and thus the faster the degradation of the material.

Bovine Achilles tendon collagen (Type I, insoluble) from the Sigma Chemical Company was used as a standard. It gives a known activity of 202 enzyme units. By definition (Sigma 1977), one enzyme unit "will liberate Amino Acids from Collagen equivalent in Ninhydrin color to 1.0 Mole of L-Leucine in 5 hours at pH 7.4 at 37° C."

Unprocessed 20-mesh milled bovine collagen (the major raw material in the manufacture artificial skin) was used as a control. When the enzyme units of a day's assay were calculated and normalized to the Sigma standard of 202, the bovine collagen was expected to yield enzyme units in the range of 178 + 15.

The following summarizes the steps in the collagenase assay.

1. For each collagen type, weigh out 0.025 g of collagen into each of 4 test tubes; one tube is the spectrophotometric blank.

2. Incubate each test sample in 5 ml of a pH 7.4 buffer with 0.10 ml of a buffered solution of 0.08% w/v collagenase (0.10 ml deionized water in each blank) for 5 hours at 37° C; stir continuously by using a magnetic flea in each tube. 3. Filter the contents of each tube to eliminate turbidity that may interfere with the spectrophotometric measurements; save 0.20 ml of each filtrate. 4. Add 2 ml of a pH 5.5 ninhydrin-and-hydrindantin solution to stop the enzymatic reaction and to induce color. 5. Place the tubes in a boiling water bath for 20 minutes.

6. Mix in 10 ml of 50% propanol, or 5 ml for highly crosslinked materials; let stand 15 minutes at room temperature to develop and fix color.

7. Record transmittance at 600 nm. Steps 4 through 7 were also done on a calibration tube containing 0.20 ml of 0.002 M L-leucine and on a corresponding blank containing 0.20 ml of deionized water.

The enzyme units of each test (3 test tubes per collagen type) were calculated from the transmittances and by comparison to the calibration test indicating the transmittance of a known amount of L-Leucine. The mean standard deviation of each set of three results was determined. Finally, these numbers were multiplied by the factor which normalized the mean enzyme units for Sigma collagen to 202.

EQUIVALENTS

While this invention has been particularly shown and described with references to preferred embodiments thereof, it will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention as defined by the appended claims. Those skilled in the art will recognize or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described specifically herein. Such equivalents are intended to be encompassed in the scope of the claims.

Claims

CLAIMSWhat is claimed is:

1. A porous biodegradable microparticle for tissue regeneration at an internal wound site in a subject, wherein: a) the pores of said microparticle have an average diameter of between about 1 ╬╝m and about 300 ╬╝m; b) the microparticle has a minimum water content of at least about

80% ; a minimum specific surface area of at least about 10³ mm² per cm^3; and an average diameter from about 10 ╬╝m to about 1000 ╬╝m; c) between about 20% to about 80% by weight of the the microparticle is biodegraded at the wound site during the time period required for a wound of about the same severity, size and tissue type to complete about one half of the contraction which normally takes place in the absence of the microparticle; and d) the microparticle comprises: i) a three dimensional network of polymers which is substantially insoluble under physiological conditions; and ii) one or more specific cell-binding fragments.

2. The microparticle of Claim 1 wherein: a) the microparticle has a minimum water content of at least about

95%; and b) between about 40% to about 60% by weight of the microparticle is biodegraded at the wound site during the time period required for a wound of about the same severity, size and tissue type to complete about one half of the contraction which normally takes place in the absence of the microparticle.

3. The microparticle of Claim 2 wherein the microparticle is suspended in a physiologically acceptable solution.

4. The microparticle of Claim 3 wherein the polymers are collagen, glycosaminoglycan, proteoglycans, fibronectin, laminin, merosine or chondronectin.

5. The microparticle of Claim 4 wherein the polymers are crosslinked.

6. The microparticle of Claim 5 wherein the specific cell-binding fragment is the specific fibronection-binding region of collagen, a specific cell-binding region of fibronectin or a proteoglycan, glycosaminoglycan or combinations thereof.

7. The microparticle of Claim 4 wherein the polymers are crosslinked collagen covalently bonded with glycosaminoglycan (GAG).

8. The microparticle of Claim 7 wherein the average molecular weight between crosslinks is about 10 kD_a to about 40 kD_a and the amount of bonded GAG is between about 0.1 to about 20 weight percent.

9. The microparticles of Claim 3 wherein the polymers are a polymer or co- polymer comprising monomer units selected from a lactone, an ╬▒-hydroxy acid or a cyclic dimer of an ╬▒-hydroxy acid.

10. The microparticles of Claim 9 wherein the ╬▒-hydroxy acid is lactic acid or gly colic acid, the cyclic dimer of an ╬▒-hydroxy acid is a cyclic dimer of lactic acid or gly colic acid and the lactone is e-caprolactone.

11. A method of promoting tissue regeneration at an internal wound site, said method comprising applying porous biodegradable microparticles to said wound site after injury, wherein: a) the pores of said microparticles have an average diameter of between about 1 ╬╝m and about 300 ╬╝m; b) the microparticles have a minimum water content of at least about 80%; a minimum specific surface area of at least about 10³ mm² per cm^3; and an average diameter from about 10 ╬╝m to about 1000 ╬╝M; c) between about 20% to about 80% by weight of the the microparticles is biodegraded at the wound site during the time period required for a wound of about the same severity, size and tissue type to complete about one half of the contraction which normally takes place in the absence of the microparticles; and d) the microparticles comprise: i) a three dimensional network of polymers which is substantially insoluble under physiological conditions; and ii) one or more specific cell-binding fragments.

12. The method of Claim 11 wherein the microparticle is suspended in a physiologically acceptable solution.

13. The method of Claim 12 wherein the microparticles are delivered to the wound site endoscopically or by laparoscopy.

14. The method of Claim 12 wherein the polymers are collagen, glycosaminoglycan, proteoglycans, fibronectin, laminin, merosine, chondronectin or combinations thereof.

15. The method of Claim 14 wherein the polymers are crosslinked.

16. The method of Claim 15 wherein the specific cell-binding fragment is the specific fibronection-binding region of collagen, a specific cell-binding region of fibronectin or a proteoglycan or glycosaminoglycan.

17. The method of Claim 14 wherein the polymers are crosslinked collagen covalently bonded with glycosaminoglycan (GAG).

18. The method of Claim 17 wherein the average molecular weight between ccrroosssslliinnkkss iiss aabboouutt 1100 kkDD_aa ttoo aabboouutt 4400 _kkaa aanndd t the amount of bonded GAG is between about 0.1 to about 20 weight percent

19. The method of Claim 13 wherein the polymers are a polymer or co- polymer comprising monomer units selected from a lactone, an ╬▒-hydroxy acid or a cyclic dimer of an ╬▒-hydroxy acid.

20. The method of Claim 19 wherein the ╬▒-hydroxy acid is lactic acid or glycolic acid, the cyclic dimer of an ╬▒-hydroxy acid is a cyclic dimer of lactic acid or glycolic acid and the lactone is e-caprolactone.

21. A method of promoting tissue regeneration at a wound site on an internal organ, said method comprising applying a porous biodegradable implant to said wound site after injury, wherein: a) the pores of said implant have an average diameter of between about 1 ╬╝m and about 300 ╬╝m; b) the implant has a minimum water content of at least about 80% ; and a minimum specific surface area of at least about 10³ mm² per cm³; c) between about 20% to about 80% by weight of the implant is biodegraded at the wound site during the time period required for a wound of about the same severity, size and tissue type to complete about one half of the contraction which normally takes place in the absence of the implant; and d) the implant comprises: i) a three dimensional network of polymers which is substantially insoluble under physiological conditions; and ii) one or more specific cell-binding fragments.

22. The method of Claim 21 wherein: a) the implant has a minimum water content of at least about 95 % ; and b) between about 40% to about 60% by weight of the implant is biodegraded at the wound site during the time period required for a wound of about the same severity, size and tissue type to complete about one half of the contraction which normally takes place in the absence of the implant.

23. The method of Claim 22 wherein the polymers are collagen, glycosaminoglycan, proteoglycans, fibronectin, laminin, merosine, chondronectin or combinations thereof.

24. The method of Claim 23 wherein the polymers are crosslinked.

25. The method of Claim 24 wherein the specific cell-binding fragment is the specific fibronection-binding region of collagen, a specific cell-binding region of fibronectin or a proteoglycan or glycosaminoglycan.

26. The method of Claim 22 wherein the polymers are crosslinked collagen covalently bonded with glycosaminoglycan (GAG).

27. The method of Claim 26 wherein the average molecular weight between crosslinks is about 10 kD_a to about 40 kD_a and the amount of bonded GAG is between about 0.1 to about 20 weight percent.

28. The method of Claim 22 wherein the polymers are a polymer or copolymer comprising monomer units selected from a lactone, an ╬▒-hydroxy acid or a cyclic dimer of an ╬▒-hydroxy acid.

29. The method of Claim 28 wherein the ╬▒-hydroxy acid is lactic acid and glycolic acid, the cyclic dimer of an ╬▒-hydroxy acid is a cyclic dimer of lactic acid or glycolic acid and the lactone is e-caprolactone.

30. A method of promoting tissue regeneration at an internal wound site in a subject, said method comprising binding cells which induce wound contraction at the wound site to a template which is substantially insoluble in physiological solution, said binding beginning after injury.

31. The method of Claim 30 wherein said binding is continued for a duration corresponding to the period of time required to complete about one half of the contraction in an untreated wound of about the same severity, size and tissue type as the wound being treated in the subject.

32. A method of promoting tissue regeneration at an internal wound site in a subject, said method comprising removing cells which induce wound contraction from the wound site, said cells being removed after injury.

33. The method of Claim 32 wherein said removal is continued for a duration corresponding to the period of time required to complete about one half of the contraction in an untreated wound of about the same severity, size and tissue type as the wound being treated in the subject.