CA2285161A1 - In vitro cultured ligament tissue and method of making same - Google Patents

Abstract

An implantable tendon-like or ligament-like tissue construct involving the creation of a tissue from a composite of a donor or source scaffold derived from allograft, autograft or xenograft of collagen fibers and directly seeded ligament or tendon fibroblasts. The fibroblasts are host derived, expanded in vitro, and are seeded directly on the scaffold.
Following initial seeding, secondary collagen seeding is performed around the scaffold, which allows the delivery of high numbers of fibroblasts and sufficient collagen for these cells to rapidly produce a ligament or tendon-like tissue material in vitro. The construct is cultured in vitro for a sufficient period of time to allow cell mediated matrix organization and integration, after which the construct may be implanted at the desired host site. Most preferably, a rat tail tendon is selected as the source xenograft for the collagen fiber scaffold.

Description

IN VITRO CULTURED LIGAMENT TISSUE AND METHOD OF MAKING SAME
SCOPE OF THE INVENTION
The present invention relates to a ligament tissue construct and a method of making a ligament tissue construct in vitro for implantation in a human or other animal subject.
BACKGROUND OF THE INVENTION
Ligament tissues perform bone-to-bone connection in humans and other animals and most typically comprise a dense band of connective tissue which is primarily composed of the protein collagen. Ligament injuries which occur when the connective tissues tear or detach from bone completely are common and frequently do not heal well. For example, the injury to the ligaments of the knee, and in particular the anterior cruciate ligament (ACL), has been the subject of considerable research as ACL injuries often result in joint instability and the subsequent onset of osteoarthritis.
Current methods of ACL reconstruction involve a surgical autograft procedure in which a portion of the patient's patellar tendon or quadriceps tendon is harvested and implanted at the knee joint. It has been found, however, that although initially strong, the tendon autograft tends to remodel over time and is replaced by weaker scar tissue which is subject to fatigue failure and creep, leading to laxity in the joint. Remodeled autografts are weaker than the original ligament and are therefore susceptible to reinjury.
The use of tendon or ligament xenografts and allografts avoids the healing problems associated with harvesting autograft tendons. Ligament replacement with xenograft and allograft implants, however, introduce the problems associated with the potential rejection of the donated graft as well as possible disease transmission. Often supplies of allogeneic material may be limited, and a significant antigenic response is associated with the implantation of unprocessed allogeneic material. Xenografts may be more readily available, however, it is typically more antigenic than allograft implants. Even if the antigenic problems and viral/prion transmission threat associated with allogeneic and xenogeneic implants are neutralized through chemical processing, the problem of in vivo remodeling remains and weakened scar tissues may form in the same manner as within autogenous implant material.
In an effort to produce better graft materials, biodegradable polymers, acellular and chemically-processed biological materials and reconstituted chemically-crosslinked collagen matrices have been utilized. Although all these materials exhibit good initial strength, the cells that repopulate these grafts typically are not ACL specific fibroblasts and as a result, they suffer the same loss of strength as autografts. Furthermore, as polymers degrade, they can incite a giant cell (macrophage) reaction.
SUMMARY OF THE INVENTION
It is an object of the present invention to provide an engineered tissue for use in replacing damaged ligament and/or tendon tissues which mirrors as closely as possible the original tissues in terms of function, structure and composition.
Another object of the invention is to provide a method of forming tendon-like or ligament-like tissues in vitro for later implantation into a human or animal subject.
A further object of the invention is to provide an in vitro cultured tendon-like or ligament-like tissue from source allogeneic or xenogeneic material, and which has been seeded with fibroblasts from the site of intended use.
The present approach to the in vitro formation of an implantable tendon-like or ligament-like tissue construct involves the creation of a ligament from a composite of a donor or source scaffold derived from allogeneic, autogeneic or most preferably xenogeneic collagen fibers and directly seeded ligament or tendon fibroblasts. More preferably, the seeded fibroblasts are host derived, expanded in vitro, and are seeded directly on a scaffold, such as detergent extracted xenogeneic tendon collagen fibers, polymers or other suitable biodegradable materials. Most preferably, following initial seeding, secondary collagen seeding is performed around the scaffold. Secondary, collagen seeding allows the efficient delivery of high numbers of host site fibroblasts and sufficient collagen for these cells to rapidly produce a ligament/tendon tissue-like material in vitro. Optionally, endothelial cells (host derived), peptides, growth factors, cytokines are also added with the collagen and cells during secondary seeding. The construct is cultured in vitro for a sufficient period of time to allow cell mediated matrix organization and integration, after which the construct may be implanted at the desired host site.
In a preferred embodiment, a rat tail tendon is selected as the source xenograft for the collagen fiber scaffold. The primary function of the scaffold material is to provide high strength during the first six to twelve months after implantation, and in that regard it is similar to a patellar autograft. The rat tail tendon core is composed of groups of substantially aligned, elongated tendon fibers, and the seeding of the rat tail tendon with fibroblasts from the implantation site is performed so as to maximize fibroblast attachment throughout the tendon fibers. The fibroblast cells improve tendon-collagen integration.
Although not essential, the tendon or ligament construct may be contiguous at one or each end with a disc, plug and/or pin formed from an implantable material.
Suitable implantable materials would include titanium or stainless steel, as well as biomaterials such as porous condensed calcium polyphosphate (CPP), as is disclosed in Canadian Patent Application No.
2,252,860, filed 15 May 1997, and laid open to the public December 4, 1997.
Optionally, cyclic or continuous loading can be used during the in vitro culturing of the ligament-type tissue, to stimulate cell mediated matrix organization and crosslinking.
BRIEF DESCRIPTION OF THE DRAWINGS
Reference is now made to the following detailed description, taken together with the accompanying drawings in which:
Fig. 1 is the appearance of a ligament-like tissue construct in culture and formed in accordance with a preferred embodiment of the invention;

Fig. 2 is a photomicrograph of an engineered ligament tissue grown in accordance with the present invention under four weeks of constant tension; and Fig. 3 is a photomicrograph of an engineered ligament tissue grown in accordance with the present invention under two weeks of constant tension interrupted with daily periods of cyclic tension.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
The invention as shown in Figure 1 relates to a ligament-like construct 10 which has been prepared in vitro for later implantation into a human or animal host subject as either a ligament and/or tendon tissue.
The construct 10 consists of an engineered fibrous ligament-like tissue 12 which is contiguous at each of its ends with an implantable plug 14a,14b. As will be described hereafter, the engineered ligament-like tissue 12 is formed in vitro by seeding and culturing ligament or tendon fibroblast cells in collagen material surrounding a suitable scaffold.
The plugs 14a,14b are formed from a porous condensed calcium polyphosphate (CCP) material, such as that described in Canadian Patent Application No. 2,252,860 to facilitate bone growth therein. The engineered ligament tissues 12 may be mechanically attached to each plug 14a,14b or alternately, may be cultured directly thereon. The design of the biodegradablelbioabsorbable CPP facilitates osteoblast ingrowth and it will become replaced with bone over time.
To insert the construct 10 into a host patient, a bore corresponding in diameter to each plug l4a,l4b is formed in each of the patient's bones to be joined by construct. The construct 10 is then positioned in place by fitting in each plug into the complementary bores in a pressure fit manner.
(a) Scaffold Preparation In the in vitro preparation of the engineered ligament tissue 12, a source xenogeneic tendon is initially obtained to serve as the framework or scaffold for the cultured ligament.

Preferably, the source xenograft is selected from tendon tissues characterized by spaghetti-like collagen fibers which can be extracted as an elongated bundle and readily separated from each other.
The tails of rats have been found to be particularly suitable as providing a suitable three dimensional scaffold material. The tendon in rat tail is composed of numerous very long fibers each with a diameter of approximately 100 ~m and, unlike most tendons, these fibers are physically distinct and easily separated. Bundles of rat tail fibers are very strong with maximum strength comparable to that of ligaments. In addition, mechanical testing on rat tail tendon samples reveals that their mechanical properties compare well with that of human anterior cruciate ligament. As previously indicated bundles of tendon fibers can be combined to match the dimensions and strength of the tissue it would replace. For example, to match the properties of the human ACL, which has an ultimate tensile load of 2195 N and stiffness of 306 N (Am. J.
Sports Med 25, 472-478, 1997), the diameter of and length of the tendon construct would need to be 7mm and 75mm respectively. A tendon bundle with a diameter of 3-4 mm would provide initial strength to that of traditional grafts used in the tendon and ligament repair (381-678 N).
The rat tail fibers are composed primarily of type I collagen, a highly conserved protein that differs little from the type I collagen that makes up the bulk of human ACL.
Fibroblasts attach rapidly to type I collagen and grow in contact with this protein. Although rat tail tendon will be implanted as a modified xenograft as will be described, the potential immunogenicity of the graft is first mitigated through detergent extraction and coating with allogeneic or autogeneic collagen and eventually autogeneic fibroblasts. Moreover, the rat-tail tendon scaffold is advantageously biodegradable and will eventually be replaced in vivo with ligamentous tissue.
In use, the rat tail is severed at its base following euthanasia of the rat.
The tail is then frozen and thawed three times to kill and lyse the tendon cells, after which the tail is immersed in a 4°C bath consisting of 70% ethanol for a minimum of 20 minutes to kill any bacteria. The tendon xenograft is then physically extracted from the tail by pulling longitudinally with hemostats. The extracted tendon xenograft has a length of between about 2 and 15 cm, and most preferably about 10 cm, possessing a spaghetti-like fiberous construction in which each of the tendon fibers are elongated and generally parallel to each other.

Following the physical extraction of the source tendon xenograft, the tendon fibers are again immersed in a 70% ethanol bath at room temperature for a period of approximately 30 minutes, to kill most of the remaining contaminating bacteria and reduce the lipid content of the tendon.
The tendon fibers are thereafter cleaned by washing sequentially ten times in a beaker containing 100-250 ml of an aqueous buffered solution having a pH selected between 6.5 and 8.
Preferred solutions include phosphate buffered saline solution (PBS) which is magnesium and calcium-free. The tendon fibers are maintained in the PBS bath for a period of 30 minutes and at a temperature of between about 20 and 25°C. The immersion of the tendon fibers in the phosphate buffered saline solution (PBS) removes further cellular debris and blood. The absence of Mg2+/Ca2+ further inactivates some proteases.
Further cleaning is next performed by immersing the tendon fibers in a cleansing bath of 250-500 ml of a water and non-ionic detergent solution. Suitable solutions would therefore include those containing 0.1 % Triton-X 100TM detergent, and the solution is maintained at room temperature (20-25°C) and changed three times over a 24 hour period. On immersion in the solution, the tendon fibers optionally may be gently agitated to remove non-collagenous proteins, some lipids, as well as antigenic elements from within the fiber bundle.
After cleansing in the 0.1 % Triton-X 100TM detergent solution, the tendon fibers are incubated for 72 hours in 250-500 ml of a 0.1 % ionic detergent solution of sodium dodecyl sulfate (SDS). The SDS solution is kept at room temperature and changed three times over a 72 hour period. Incubation in the SDS solution removes any additional antigenic elements.
The extracted tendon fibers are thereafter washed 10 times with 250-500 ml water over 1 hour followed by 10 changes of 250-500 ml purified water over 24 hours at room temperature to remove any residual detergent. Histologically the tendon appears acellular after this extraction procedure. Strength is not significantly affected. Higher concentrations of either detergent, although possible, disadvantageously disrupt tendon collagen organization and reduce the strength of the tendon.
(b) Initial Collagen Seeding Tendon fibers are organized into bundles of appropriate diameter to match that of ligament and provide sufficient strength. Preferably, the ends of the bundles are tied at their ends to maintain the generally longitudinal orientation. Preferably the tendon fibers are held in longitudinal tension and the ends of the tendon fibers are tied off, crimped or otherwise secured to maintain the generally longitudinal orientation of the individual fibers.
The bundles of tendon fibers form a scaffold for ligament cell growth and serve as a very strong stint following implantation. The individual tendon fiber diameter ( 100 pm) and composition are ideal for ligament fibroblast attachment. It is expected that the rat tail tendon fibers will remodel overtime and will be replaced with new collagen over a period of time after implantation.
The tendon scaffold is next again sterilized, as for example, by incubation in 70% ethanol for 30 minutes with mixing (21°C) followed by incubation in lOx antibiotic for 24 hours (37°C in tissue culture incubator). The sterility of the scaffold is then confirmed by swabbing or incubation in medium without antibiotic for 48 hours (37°C in tissue culture incubator supplemented with 5% C02 and high humidity).
The scaffold tendon fibers are then positioned within a seeding trough and directly seeded with ligament fibroblasts. Although not essential, the initial seeding is preferably performed with the scaffold longitudinally stretched in a trough and medium with seeding fibroblast cells which have been harvested from the host site at which the construct is to be implanted is added. The medium transferred to the seeding trough contains ligament fibroblasts (0.5 to 10 x 106 cells) and is seeded directly onto a bundle of tendon fibers in minimal volume of medium, such as DME (Dulbecco's Modified Eagle's Medium). The spaghetti-like fibers of the scaffold are physically separated during initial collagen seeding to permit the fibroblast cells to penetrate throughout the scaffold interior. The result is that fibroblast attachment is not merely restricted to the outer periphery of the scaffold, but extends through its entirety.

The fibroblasts are most preferably ligament specific and expanded in vitro.
Direct seeding of tendon fibers ensures ligament fibroblast infiltration into the tendon fiber bundle and facilitates collagen seeded fibroblast layer integration. Cells are allowed to attach and proliferate for 2-3 days (37°C in tissue culture incubator, supplemented with 5%
COz and high humidity) while periodically topping up the seeding medium.
The seeded scaffold 16 (Figure 1 ) is moved directly from the trough and secured under constant low tension in an axially vertical position within a vertically oriented cylindrical tube.
The seeded scaffold is grown for a further 3 to 4 days to allow matrix production and cell proliferation prior to secondary fibroblast seeding in a collagen solution.
(c) Secondary Seeding Following initial fibroblast seeding and growth on the seeded scaffold 16, secondary seeding is performed wherein the scaffold 16 is seeded with ligament specific fibroblasts combined with purified type I collagen. Sterile acid or pepsin purified collagen dissolved in acetic acid (pH 3.0) 1mM HC1 (4°C) is added to fibroblasts suspended in a suitable medium (37°C) Dulbecco's Modified Eagle's medium (DME) supplemented with 15%
fetal bovine serum and ascorbic acid (10-100 ug/ml). The medium concentration is adjusted for the diluting effect of the collagen solution and then the pH is adjusted to 7.2 with NaOH. The final collagen concentration ranges from 0.6-1.0 mg/ml, and final cell concentration ranges from 0.8-3 x 105 cells/ml, although the total amount of collagen and cell number depends on the size of tissue to be formed.
The tube is filled (20 to 90% by volume) with the hydrated collagen and cell solution and incubated at 37°C. The mixture polymerizes around the seeded scaffold 16 very rapidly at 37°C.
The collagenous matrix 18 is contracted by the fibroblasts around the seeded tendon scaffold.
The cellular matrix shrinks radially and longitudinally towards the scaffold, forming a construct having a generally elongated cylindrical configuration. After 1 to 4 weeks of culture in vitro with feeding as needed, the cells have reorganized and remodeled the collagen matrix into a ligament-like tissue 12.
Remodeling of the collagen matrix by ligament fibroblasts is preferably achieved with the tissue grown under tension. Constant tension is sufficient to induce matrix reorganization. This organization is, however, more rapid when the tissue is subjected to periods of cyclic tension with or without constant tension (lHz, 1800-36000 cycles/day). Figures 2 and 3 show the effect of the application of cyclic tension. Figure 2 represents the ligament-like tissue following culturing under constant tension for 4 weeks. Figure 3 shows the ligament-like tissue following culturing for 2 weeks under constant tension with daily periods of cyclic tension at 1 Hz, 1800 cycles/day. Figure 3 shows enhanced organization of the fibroblasts and increased alignment of collagen fibers achieved with cyclic tension over a shorter culturing time.
Some crimping of the collagen fibers, more closely resembling undamaged ligaments, may also be seen.
The shape of this tissue can be modified by changing the shape of the chamber used during secondary collagen seeding and/or by the use of internal anchors or structures within the chamber. In particular, the tube shape may be altered to form a ligament construct having a desired profile, or the anchors could be employed within the tube to secure the engineered tissue into a desired shape. For example, where a generally cylindrical ligament implant is desired, secondary seeding of the scaffold is performed in a generally cylindrical tube. Where a flatter ligament construct is desired, the secondary seeding may occur in an elongated thin rectangular tube. Similarly, the overall dimensions of the tube may be adjusted depending upon the amount of collagen and number of cells which are to be delivered and on the size of the ligament to be constructed.
Secondary collagen seeding of the tendon scaffold ensures a known number of ligament fibroblasts are delivered to the engineered tissue, as contrasted with direct seeding which fails to ensure that all seeded cells attach to the scaffold and where a substantial proportion may fall through the spaces between the fibers. The presence of sufficient cell numbers around the scaffold ensures adequate in vitro and in vivo remodeling. Where a larger number of cells are to be seeded, larger tubes, a larger volume of hydrated collagen material, and longer incubation times are used to perform secondary seeding functions.
In addition to acting as an efficient way to deliver a large and known number of cells to the scaffold, collagen seeding also provides the engineered tissue with a substantial amount of collagen which can be reorganized into ligament-like matrix. This protein makes up approximately 80% of the dry weight of a ligament. The number of fibroblasts normally used to collagen seed would not be able to synthesize the equivalent amount of collagen during the same culture period in vitro.
While the preferred embodiment discloses the use of CCP plugs 14a,14b, it is to be appreciated that the plugs could be omitted in their entirety and the engineered ligament 12 implanted directly. Alternatively, other implantable structures including by way of example staples, pins, screws and the like could also be used. The plugs 14a,14b may be formed from any biologically suitable materials, including by way of non-limiting examples, metals, resins, minerals and plastics, which will now become apparent to persons skilled in the art.
Although the preferred embodiment of the invention describes and illustrates rat tail tendons as being a suitable scaffolding structure, it is to be appreciated that other types of tendons and tissues may also be used with the present invention including by way of non-limiting example, tail tendons from other animals of the order R ti or Marsupialia.
Although not essential, source tendons would preferably also have a similar fibrous structure to permit cell attachment and/or movement into the interior of the tendon tissue bundle.
Reconstituted collagen fibers may also be potentially suitable but are not as strong and highly cross-linked.
Although the detailed description describes the preferred embodiment in the formation of a ligament-like tissue, it is to be appreciated that tendon-like tissues could be formed in a similar manner.

While the detailed description describes various preferred embodiments, the invention is not so limited. Many modifications and variations will now occur to persons skilled in the art.
For a more precise definition of the invention, reference may be had to the appended claims.

Claims (17)

1. A method of forming an implantable tissue in vitro by the steps of, selecting a source tendon, substantially removing cellular debris from said source tendon to form a collagen fiber scaffold, initially seeding said scaffold with fibroblasts, and culturing said seeded scaffold to permit cell mediated matrix organization.

2. The method of claim 1 wherein said cellular debris is removed by immersing said tendon in a disinfecting solution, followed by immersion in an aqueous buffered solution.

3. The method of claim 1 wherein said source tendon comprises a plurality of generally parallel elongated collagen fiber strands.

4. The method of claim 1 wherein during said culturing step, periodically maintaining the scaffold under variable tension.

5. The method of claim 1 wherein during said culturing step, periodically maintaining the scaffold under substantially constant tension.

6. The method of claim 1 comprising the further step of secondary seeding said scaffold with a said hydrated collagen matrix comprising type 1 collagen and fibroblasts selected from either tendon fibroblasts or ligament fibroblasts.

7. The method of claim 2 wherein said disinfecting solution comprises an alcohol.

8. The method of claim 3 wherein said source tendon comprises a rat tail tendon.

9. The method of claim 6 wherein said scaffold is secondary seeded by immersing said scaffold in a hydrated purified collagen matrix populated with said fibroblasts.

10. The method of claim 3 wherein during said initial seeding step, separating said collagen fiber strands to permit attachment and/or movement of said fibroblasts into an interior of said scaffold.

11. The method of claim 6 wherein secondary seeding is performed by maintaining said scaffold in suspension in a vessel containing said hydrated collagen matrix.

12. A ligament-type tissue for implantation in humans or animals, formed by the method of claim 1.

13. A tendon-type tissue for implantation in humans or animals, formed by the method of claim 1.

14. Use of a ligament-type tissue formed by the method of claim 1 for implantation in vitro in a human or animal subject.

15. Use of a ligament-type tissue as claimed in claim 12 wherein said seeded fibroblasts are collected from said human or animal subject.

16. Use of a tendon-type tissue formed by the method of claim 1 for implantation in vitro in a human or animal subject.

17. Use of a tendon-type tissue as claimed in claim 12 wherein said seeded fibroblasts are collected from said human or animal subject.

An implantable tendon-like or ligament-like tissue construct involving the creation of a tissue from a composite of a donor or source scaffold derived from allograft, autograft or xenograft of collagen fibers and directly seeded ligament or tendon fibroblasts. The fibroblasts are host derived, expanded in vitro, and are seeded directly on the scaffold.
Following initial seeding, secondary collagen seeding is performed around the scaffold, which allows the delivery of high numbers of fibroblasts and sufficient collagen for these cells to rapidly produce a ligament or tendon-like tissue material in vitro. The construct is cultured in vitro for a sufficient period of time to allow cell mediated matrix organization and integration, after which the construct may be implanted at the desired host site. Most preferably, a rat tail tendon is selected as the source xenograft for the collagen fiber scaffold.