WO2006110110A1

WO2006110110A1 - Tissue construct and method thereof

Info

Publication number: WO2006110110A1
Application number: PCT/SG2006/000092
Authority: WO
Inventors: Hongwei Ouyang; Cho Hong James Goh; Siew Lok Toh; Tong Earn Tay
Original assignee: National University Of Singapore
Priority date: 2005-04-11
Filing date: 2006-04-10
Publication date: 2006-10-19
Also published as: WO2006110110A8

Abstract

There is provided a tissue construct comprising at least one structure and multipotent cells and a method for the preparation of the tissue construct. The multipotent cells may be in the form of a sheet before assembly with the at least one structure. The tissue construct may be used as a graft, particularly as a graft for connective tissue.

Description

Tissue construct and method thereof

Field of the Invention

The present invention is in the field of tissue engineering. The invention generally relates to tissue constructs and to method(s) for the preparation thereof. Such tissue constructs may be used as artificial grafts, for example, to replace or supplement natural tissue or parts of organs.

Background of the Invention

Clinical use of grafts of living tissues has recently been moved from direct implantation of freshly harvested fully formed tissue to tissue engineering strategies involving seeding of cells on scaffolds (also variously referred to as substrates, supports or matrices) which will regenerate or promote the regeneration of local structure. In this strategy, cell seeding and mass transports of nutrients and waste matter produced are two of the important issues for successful tissue engineering. Current cell seeding techniques include: (1) delivering cell-gel composite into structure; and (2) delivering cell suspension into structure in static or dynamic situation.

However, there are some disadvantages in the above current techniques. In the cell-gel composite delivery technique, cells can be immobilized in the structure completely. However, it raises the issue of mass transport because the gel will fill up the pores in the structure. It is known that oxygen is effectively depleted if the space within the tissue is about 100-200 microns apart, which in turn will significantly affect the pH values (Colton et al, 1995). It has been observed that cell necrosis in fibrin gel increases with the distance from surface of the structure. On the other hand, if the internal void volume of structure for fibrovascular ingrowth is occupied by gel material, vascularization of the engineered tissues will be delayed which will then lead to slow tissue repair and regeneration.

In the cell suspension delivery technique, the distribution and attachment of cells in sponge or fiber structures are mainly determined by gravity (Sittinger et al, 1996). Although the cell attachment of fiber structures can be improved by different pre-coating techniques and modifying the porosity and surface charge of the structure, 30^40% of the initially seeded cells do not attach to the structure, thus it will hinder further tissue development (Zund et al, 1999). Under a given structure geometry and porosity, increasing the cell attachment surface of the structure would decrease the pore size. However, large pore size is essential for promoting host fibrovascular in-growth into the structure (Mikos et al, 1993).

The use of the above current cell seeding techniques has posed some conflicting goals. These are: (a) In designing the structures, it is preferred to have high porosity so that more cells can attach to the surfaces. However, high porosity will mean low mechanical strength of the structures.

(b) To promote larger surface area for cell attachment, more structure materials are required which in turn, will increase the inflammatory reaction.

The use of the cell sheet technique for tissue engineering has been attempted by several researchers. McAllister et al (2003) invented a novel approach to tissue-engineered blood vessel (TEBV) production that is based exclusively on the use of cultured human cells, i.e., without any synthetic or exogenous biomaterials. Human vascular smooth muscle cells (SMC) were cultured to produce a cohesive cellular sheet. This sheet was placed around a tubular support to produce the media of the vessel. A similar sheet of human fibroblasts was wrapped around the media to provide the adventitia. Pouliot et al (2002) did an in-vitro reconstruction of an autologous skin made of fibroblast sheet and devoid of exogenous extracellular matrix proteins and synthetic material. The absence of synthetic materials precluded foreign body reactions, thus increasing the likelihood of a long-term graft success. However, these cells normally take a very long time (4 to 8 weeks) to form cell sheets. Moreover, the mechanical strength of the cell sheets is too weak to withstand the physical loads. They are not suitable for tissue regeneration of bone, tendon/ligament and cartilage. These drawbacks limit the use of the cell sheet technique for tissue engineering applications and researchers have to continue the use of conventional methods such as cell suspension or cell-gel seeding to apply cells to tissue engineering structures.

Thus, some of the problems in the tissue grafts of the current art, particularly those to replace connective tissue, include weak cell adhesion to scaffold, lack of mechanical strength, low cell seeding, low mass transport of nutrients and waste products, and lack of in-growth by host cells.

There is therefore a need in the art for improved and suitable tissue constructs for use as grafts in various parts of the body.

Summary of the Invention

The present invention addresses the problems of the art and provides a novel tissue construct and a method thereof.

According in one aspect, the present invention provides a tissue construct comprising at least one structure and multipotent cells. The multipotent cells are in the form of a sheet before assembly with the at least one structure. In particular, the structure is a structure of biocompatible material. The multipotent cells may comprise mesenchymal stem cells. The mesenchymal stem cells may be adult and/or embryonic stem cells. The mesenchymal stem cells may be isolated from autologous and/or allogenic sources. The tissue construct may further comprise at least one therapeutic agent. The at least one therapeutic agent may be selected from the group consisting of angiogenic growth factors, angiogenic inhibitors, growth factors, thrombin inhibitors, antithrombogenic agents, thrombolytic agents, fibrinolytic agents, vasospasm inhibitors, calcium channel blockers, vasodilators, antihypertensive agents, antimicrobial agents, antibiotics, inhibitors of surface glycoprotein receptors, anti-platelet agents, antimitotics, microtubule inhibitors, anti-secretory agents, actin inhibitors, remodeling inhibitors, antisense nucleotides, antimetabolites, antiproliferatives, anticancer chemotherapeutic agents, antiinflammatory steroid or non-steroidal anti-inflammatory agents, immunosuppressive agents, growth hormone antagonists, growth factors, dopamine agonists, radiotherapeutic agents, peptides, proteins, enzymes, extracellular matrix components, inhibitors, free radical scavengers, chelators, antioxidants, anti-polymerases, antiviral agents, photodynamic therapy agents, and gene therapy agents.

The structure of the tissue construct may be in the form of a sponge, foam, mesh of fibers, sheet, woven or knitted materials and the structure may comprise natural and/or synthetic materials. Where a synthetic material is used, it may be ceramic material, biosorbable and/or biodegradeable material. The biosorbable material may be poly (L-lactide) (PLLA), polyglycolide (PGA), polylactide (PLA), polycaprolactone (PCL), poly(glaxanone), poly(orthoesters), poly(pyrolicacid), poly(phosphazenes and/or their derivatives. The tissue construct may comprise at least one natural material such as collagen, gelatin, silkworm silk, spider silk, allogenic fascia tisues, autologous fascia tissues or alimentary canal/intestinal submucosa.

The tissue construct may be suitable for use as a graft, particularly for use as a graft for connective tissue or skin. In particular, the tissue construct may be used as a graft for ligaments, tendons and/or skin. The tissue construct may also be suitable for use in wound healing, urinary or gall bladder reinforcement patch or replacement, or as a vascular graft.

In another aspect, the present invention provides a kit comprising the tissue construct of the present invention. The kit may further comprise packaging and information pertaining to use of the tissue construct.

In another aspect, the present invention provides a method of preparing a tissue construct, the method comprising: providing at least one structure; preparing at least one sheet of multipotent cells, and assembling the tissue construct from the at least one structure and at least one sheet of multipotent cells. In particular, the structure is a structure of biocompatible material or comprises at least one biocompatible material.

Under the method of the present invention, the assembling may further comprise stacking, rolling, folding and/or wrapping the tissue construct. The tissue construct may be further cultured after assembly. The multipotent cells of the tissue construct may comprise mesenchymal stem cells and the mesenchymal stem cells may be adult and/or embryonic stem cells. The mesenchymal stem cells may be isolated from autologous and/or allogenic sources.

The method may further comprise adding at least one therapeutic agent to the construct. The at least one therapeutic agent may be selected from the group consisting of angiogenic growth factors, angiogenic inhibitors, growth factors, thrombin inhibitors, antithrombogenic agents, thrombolytic agents, fibrinolytic agents, vasospasm inhibitors, calcium channel blockers, vasodilators, antihypertensive agents, antimicrobial agents, antibiotics, inhibitors of surface glycoprotein receptors, anti-platelet agents, antimitotics, microtubule inhibitors, anti-secretory agents, actin inhibitors, remodeling inhibitors, antisense nucleotides, anti-metabolites, antiproliferatives, anticancer chemotherapeutic agents, anti-inflammatory steroid or non-steroidal anti-inflammatory agents, immunosuppressive agents, growth hormone antagonists, growth factors, dopamine agonists, radiotherapeutic agents, peptides, proteins, enzymes, extracellular matrix components, inhibitors, free radical scavengers, chelators, antioxidants, anti-polymerases, antiviral agents, photodynamic therapy agents, and gene therapy agents.

The method may further comprise adding at least one natural material to the construct. The natural material may be collagen, gelatin, silkworm silk, spider silk, allogenic fascia tissue, autologous fascia tissues, and alimentary canal/intestinal submucosa.

The structure of the tissue construct prepared by the method of the present invention may be in the form of a sponge, foam, mesh of fibers, sheet, knitted or woven materials and the structure may comprise natural and/or synthetic biocompatible materials. Where synthetic materials are used, they may be ceramic materials, biosorbable and/or biodegradable materials. The biosorbable materials may be poly (L-lactide) (PLLA), polyglycolide (PGA), polylactide (PLA), polycaprolactone (PCL), poly(glaxanone), poly(orthoesters), poly(pyrolicacid), poly(phosphazenes and/or their derivatives.

The tissue construct prepared by the method of the present invention may be suitable for use as a graft, particularly for use as a graft for connective tissue or skin. In particular, the tissue construct may be used as a graft for ligaments, tendons and/or skin. The tissue construct may also be suitable for use in wound healing, urinary or gall bladder reinforcement patch or replacement, or as a vascular graft.

Brief Description of the Figures

Figure 1 shows the basic components of the present invention comprising a mesenchymal stem cell sheet and a structure. In particular, the structure is a structure of biocompatible material. Though the figure shows the cell sheet and the structure in rectangular shape, they may also be in other shapes such as circles or triangles. The tissue construct is prepared by assembling the stem cell sheet and the structure.

Figure 2 shows the tissue construct in a rolled form.

Figure 3 shows the tissue construct assembled with a single fold.

Figure 4 shows the tissue construct assembled with multiple folds.

Figure 5 shows the tissue construct assembled by wrapping the stem cell sheet around the structure.

Figure 6 shows the stacking of the components to have the structure sandwiched by the stem cell sheets.

Figure 7 shows a variation of the stacking of the components to obtain multiple layers of stem cell sheets and structures.

Figure 8 shows the histology of the longitudinal sections (a, b) and transverse section (c) of the engineered ligament analog tissue construct after 4 weeks of culture (H&E staining; original magnification, (a) 4OX and (b, c) 100X

Figure 9 shows the immunohistology of the engineered ligament analog tissue constructs after 4 weeks of culture showing the tissue constructs consisting primarily of collagen type I (a) and small amount of collage type III (b) and tenascin (c). lmmunohistofluorescent staining.

Figure 10 shows histograms comparing the mean (± SD) tensile stiffness and maximum force of ligament analog tissue constructs with scaffold alone 4 weeks of culture after assembly. Figure 11 shows macromorphology (a), transmission image (b), and viable cells as indicated by positive CFDA staining under confocal microscopy of bone Marrow Stroma Cell (bMSC) sheet after assembling with knitted PLLA scaffold

(C).

Definitions

Sheet - A piece of material and/or collection of cohesive cells that is physically, chemically and/or biologically distinguishable from another piece of material and/or collection of cells. A sheet may comprise more than one type of cells and the cells may be in more than one layer of cells and/or material. Structure - A support, scaffold or substrate capable of supporting cells. The structure may be in two-dimensional form such as a sheet or it may be in three- dimensional form and possess porosity by being sponge-like, in the form of foam or having a meshwork formed by fibers or it may be made of a knitted or woven material. In particular, the structure may be a structure of biocompatible material.

Additive - Any material that is added to the structure. Examples of additives include hydroxyapatite or tricalcium phosphate to promote bone growth, and collagen and gelatin to encourage cell adhesion and/or growth. Grafting - The transplanting of a natural or artificial tissue (graft) from one place to another, for example, a piece of skin graft from a donor site to a recipient site. The transplanting of tissue can be from one part of the patient to another (autologous graft), as in the case of a skin graft using the patient's own skin; or from one patient to another (allogenic graft), as in the case of transplanting a donor kidney into a recipient.

Biosorbable, biodegradable - A material is said to be biosorbable if it can be readily broken down and absorbed by the body over a period of time. This is in contrast to implanted biodegradable materials that break down in the body over time but are not necessarily absorbed by the body. For the purpose of the present invention the term "biosorbable" may also be written as "bioresorabable" or "bioabsorbable".

Biocompatible material - Any material that will not elicit an adverse reaction in living cells or tissues such as an immunological or inflammatory response. Therapeutic agent - Any molecule, drug or compound that exerts a desired and/or beneficial effect.

Assemble - To place different components together to form an assembly or a structure. The act of assembling may entail further working (stacking, rolling, folding or wrapping) the components of the construct to assume a desired shape.

Joining of structure and/or edges - The open edges of the structure may be joined together to another edge or material by, for example, suture, clip and/or chemical means such as by a biocompatible adhesive. Edges may also be joined when the cells at the edges grown and extend to other surfaces or edges. Nanofibers - Fibers with dimensions measured in the scale of nanometers (one-billionth of a meter). By "nanometer diameter" it is meant to include fibrils ranging in diameter from approximately 200 nanometer to approximately 500 nanometers. The term "fibers" in this application refer to nanofibers. A nanofiber structure or structure is a product comprising nanofibers. The structure can further comprise cells and therapeutic agents and can be used as a structure and/or graft.

Natural materials - Materials derived from natural, non-synthetic sources such as collagen, gelatin, silk from silkworm or spider, allogenic or autologous fascia tissues, and alimentary canal/intestinal submucosa.

Detailed Description of the Invention

Biographic references mentioned in the present specification are given in the list of references at the end of the examples. The whole content of such bibliographic references is herein incorporated by reference.

The present invention provides a tissue construct and method thereof. The tissue construct comprises a sheet of cells and a structure such as a tissue engineered structure and may be useful as a graft. In particular, as a graft to supplement or replace connective tissue. The structure may be a structure of biocompatible material.

Previous studies have shown that the mesenchymal stem cells (MSCs) or bone marrow stromal cells (bMSCs) are multipotent. Multipotent cells may also be referred to pluripotent cells. They can differentiate into bone, cartilage, tendon, fat, and are able to grow well on various structures (Ouyang et al, 2002; Ouyang et al, 2003; Goh et al, 2003). Unlike other cells that display contact inhibition, the present inventors have discovered that these multipotent cells could surprisingly form cell sheets of one or more layers of cells and may thus be used in tissue engineered constructs.

The present invention aims to: to deliver the cells into structure efficiently and in a controlled manner; lessen the mass of structure and frees the design of structure from the concern of effective cell seeding; provide a large number of progenitor cells for connective tissue regeneration; preserve the matrix produced and the original cell-cell/cell-matrix interaction, thus accelerating the tissue formation after cell delivery; facilitate mass transport of nutrients and waste matter following construct assembly due to the interstitial space between cell layers; facilitate the vascularization of engineered tissue following implantation taking advantage of the interstitial space between cell layers. facilitate the vascularization of engineered tissue following implantation due to the growth factors (such as VEGF) synthesized by bone marrow derived progenitor cells; while at the same time preventing unwanted tissue in-growth and avoiding and minimzing the use of reagents for cell attachment.

Accordingly, there is provided a tissue construct comprising at least one structure and multipotent cells wherein the multipotent cells are in the form of a sheet before assembly with the at least one structure. In particular, the structure is a structure of biocompatible material. The multipotent cells may comprise mesenchymal stem cells and the the mesenchymal stem cells may be adult or embryonic stem cells. The mesenchymal stem cells may be isolated from autologous and/or allogenic sources.

To prepare the tissue construct of the present invention, there is provided a method comprising providing at least one structure; preparing at least one sheet of multipotent cells, and assembling the tissue construct from the at least one structure and at least one sheet of multipotent cells.

According to the method of the present invention, the assembling may further comprise stacking, rolling, folding and/or wrapping the tissue construct (FIGS. 3-7). The tissue construct may be further cultured after assembly. The multipotent cells of the tissue construct may comprise mesenchymal stem cells and the mesenchymal stem cells may be adult and/or embryonic stem cells. The mesenchymal stem cells may be isolated from autologous and/or allogenic sources.

The tissue construct according to the invention may be suitable for use as a graft, in particular as a connective tissue graft. More in particular, the present invention provides a three-dimensional tissue graft mimicking the host connective tissue such as ligaments, tendons, blood vessels and muscle fascia that the graft is meant to replace or supplement. The tissue construct may also be suitable for use as a skin graft, in wound healing, or as a urinary or gall bladder reinforcement patch.

Natural organs and body structures usually possess layers of cells and tissue. Each layer is usually distinct from another, such as the different muscle layers of a limb and the tendon connecting the muscle to a bone. While each layer may have a predominant type of cell, each layer may also comprise other types of cells.

In mimicking the natural tissue structure, the present invention comprises at least one structure and at least one sheet of multipotent cells. In particular, the construct of the invention may comprise at least one structure of one or more biocompatible materials. The biocompatible material or cells may be provided, for example, in the form of a layer of biocompatible material and/or a sheet of cells. The sheet of cell may have one or more layers of cells. The number of layers of biocompatible materials and/or sheets of cells depends on the application of the graft.

In one example, the present invention relates to a tissue construct for tendon and/or ligament repair. In another example, the present invention relates to a graft mimicking dermal or skin grafts

Suitable biocompatible and/or biosorbable polymer materials for use in tissue construct according to the present invention include, but are not limited to, synthetic polymers, such as poly (L-lactide) (PLLA), polyglycolide (PGA), polylactide (PLA), polycaprolactone (PCL) and their co-polymers, poly(glaxanone), poly(orthoesters), poly(pyrolicacid) and poly(phosphazenes). Other additives that can be incorporated into these materials include, but are not limited to, calcium phosphate based ceramics such as hydroxyapatite and tricalcium phosphateand natural polymers, such as collagen and gelatin. It is preferable but not necessary that the biodegradable and/or biosorbable materials be approved by food and drug authorities of specific countries for use in surgical applications. In one embodiment, the structure may be prepared by electrospinning of nanofibers.

Besides electrospun sheets, the structure of the present invention may further comprise one or more additional sheets of a biodegradable and/or biosorbable (biodegradable/biosorbable) material formed by solution casting to confer other desirable physical, chemical and/or physiological characteristics to the nanofiber structure. An example of such sheets is a solution cast sheet of PCL less than 100μm thick, that is structurally homogenous and do not possess any nanofibrillary characteristics. Solution casting of such sheets is known in the art. For example, PCL is dissolved in a suitable solvent at low concentration and poured on top a substantially flat casting tray. After the sheet has formed, residual solvent is removed in a vacuum oven. Such sheets may be further treated by addition of one or more therapeutic agents before or after assembly with the sheet of mesenchymal stem cells. The mesenchymal stem cells may be obtained directly from a suitable donor, e.g. a patient's own cells; from a culture of cells from a donor; or from established cell culture lines. Using standard cell culture techniques and conditions well-known to a person skilled in the art, the cells are grown in culture until confluent and used when needed.

Graft healing is important in determining the success or failure of a graft. All grafts, regardless of their composition and structure, evoke complex but predictable host responses. Blood-biomaterial surface reactions start immediately after restoration of circulation. The cellular and humoral responses to synthetic materials include the deposition of plasma proteins and platelets, the infiltration of neutrophils and monocytes, and the diffentiation, migration and proliferation of the mesenchymal stem cells, will determine graft failure and potential graft infection.

Another factor is that of angiogenesis. Angiogenesis is the sprouting of new capillaries from preexisting vasculature and represents a complex multistep process involving extensive interplay between cells, soluble factors, and ECM components (Steffens et al., 2004). One of the prominent shortcomings of structures for tissue engineering is their poor ability to support angiogenesis. For this reason, the structure of the present invention may be enhanced by incorporating therapeutic agents such as angiogenic growth factors, peptides or proteins. Angiogenic growth factors such as vascular endothelial growth factor (VEGF), fibroblast growth factor-1 (FGF-1), and fibroblast growth factor-2 (FGF-2), and angiogenic inhibitors such as platelet factor 4, interferons, and thrombospondin, are required to create a microenvironment in which angiogenesis is a function of balance between positive and negative regulators. Exogenous intervention such as controlled local delivery of exogenous angiogenic factors may change these balances so as to induce transmural capillary in-growth in vivo and stimulate endothelialization of prosthetic grafts.

Therapeutic agents can be incorporated into the structure by physically coating, chemically covalent-grafting or an advanced coaxial electrospinning technique (Zhang et al., 2004). In a coaxial electrospinning process, two immiscible liquid solutions are injected at appropriate flow rates through two concentrically arranged capillary tubes. At a certain range of applied electrical potential and flow rate, a structured Taylor cone is formed at the exit of the coaxial tubes with an outer meniscus surrounding the inner one. A liquid thread is issued from the vertex of each one of the two menisci, giving rise to a compound jet. After evaporation of the solvents during the course of jet flying, a "core-shell structured" bi-component composite nanofiber is produced.

Such core-shell structured nanofibers may be used to encapsulate one or more therapeutic agents by using an appropriated biodegradable polymer as a shell so that the encapsulated biomolecules may be released when the shell material degrades over time. This method provides longer term angiogenic support compared to simply coating the growth factors on the nanofibers.

Besides angiogenic growth factors, other therapeutic agents may be used in the coating. Examples of other therapeutic agents that may be used include thrombin inhibitors, antithrombogenic agents, thrombolytic agents, fibrinolytic agents, vasospasm inhibitors, calcium channel blockers, vasodilators, antihypertensive agents, antimicrobial agents, antibiotics, inhibitors of surface glycoprotein receptors, anti-platelet agents, antimitotics, microtubule inhibitors, anti-secretory agents, actin inhibitors, remodeling inhibitors, antisense nucleotides, anti-metabolites, antiproliferatives, anticancer chemotherapeutic agents, anti-inflammatory steroid or non-steroidal anti-inflammatory agents, immunosuppressive agents, growth hormone antagonists, growth factors, dopamine agonists, radiotherapeutic agents, peptides, proteins, enzymes, extracellular matrix components, inhibitors, free radical scavengers, chelators, antioxidants, anti-polymerases, antiviral agents, photodynamic therapy agents, and gene therapy agents.

The tissue construct of the present invention may comprise natural materials such as collagen, gelatin, silk from silkworm or spider, allogenic or autologous fascia tissues, and alimentary canal/intestinal submucosa to confer certain desirable characteristics such as provision of a hospital environment for the graft or host cells. Having now generally described the invention, the same will be more readily understood through reference to the following examples that are provided by way of illustration and are not intended to be limiting of the present invention.

The following non-limiting examples have been carried out to illustrate certain embodiments of the invention.

EXAMPLES

Standard tissue engineering and cell culture techniques known in the art and not specifically described are well-known in the art and may be found in reference books such as Langer et al (eds) Principles of Tissue Engineering, Elsevier Science & Technology Books (Second Edition, 2000), and Masters JRW (ed) Animal Cell Culture: A Practical Approach, Oxford University Press (Third Edition, 2000).

Example 1 - Ligament and/or tendon grafts

Method

Isolation and Culture of MSCs

Bone marrow was aspirated from the iliac crest of a 30-kg Yorkshire pig and collected into polypropylene tubes containing preservative-free heparin (1000

U/mL). The bone marrow and heparin were well mixed. Following the protocols described in Ouyang et al (2002), bone marrow stromal cells were isolated by short-term adherence to plastic as described. Nucleated cells were isolated and plated in culture medium containing Dulbecco's modified Eagle's medium (DMEM; GIBCO, Grand Island, NY)₁ fetal bovine serum (15%, w/v; HyClone, Logan, UT), penicillin (100 U/mL), and streptocycin (100 mg/mL; GIBCO). The nucleated cells were plated at a density of 5 million nucleated cells per 100-mm dish and incubated at 37°C with 5% humidified CO2. After 24 h, nonadherent cells were discarded and adherent cells were cultured. The culture medium was changed every 3 days. When the culture dishes became nearly confluent after about 14 days, the cells were detached and serially subcultured. The second- passage (P2) cells were used in this study.

Fabrication of Knitted PLLA Scaffolds

The knitted scaffold was fabricated using 3 yarns (20 filaments/yarn; diameter of filament is 25 μm) of PLLA fibers (Albany International Research Co.) on a knitting machine (Silver-reed SK270, Suzhou, China). The plain knitted PLLA scaffolds were manufactured with 6 stitches per centimeter. The pore size was 2 mm x 2 mm. The length of PLLA sheet scaffold was standardized to 30 mm x 50 mm.

Fabrication of the bMSCs Sheet/Knitted PLLA Composite Ligament Analog Tissue Construct

After P2 bMSCs were harvested, they were cultured in low glucose DMEM with 10% FBS and became confluent within 5 to 7 days. After confluence, bMSCs were cultured in high glucose DMEM with 20% FBS and 50 μg/mL ascorbic acid. Within 2 to 3 weeks a bMSC sheet was formed in a 200 cm² dish. It could be detached from the substratum by applying a small force. Then a 50-mm length of knitted scaffold was put on the cell sheet as shown in FIG. 1 and assembled by a rolling/wrapping technique to obtain the tissue construct shown in FIG. 2. The assembled cell-scaffold structure was then held in place in a 52-mm length frame device with 2 mm (4%) strain. Then the whole structure was put in a spinner flask with the speed of 6 rpm for 4 weeks and the ligament analog tissue construct thus prepared may be implanted into a recipient.

To evaluate the macromorphology, histology, and biomechanics of the tissue constructs, the PLLA ligament analog tissue constructs thus prepared and PLLA scaffolds without cells incubated with cell culture medium at the same condition were used.

Cell Labeling and Detection

CFDA molecule probe (5, 6-carboxyfluorescein diacetate) is well recognized as cell trace markers. Therefore, in this study it was used to investigate the survivability of bMSCs within the cell sheet after assembly. On the first day, the composite graft of bMSC sheet/knitted scaffold tissue construct was incubated for 20 min with 25 μM CFDA solution, and then changed with culture medium. The positive cells were observed under fluorescent microscopy on the second day. Histological and lmmunohistoflurorescent Examination

After 4 weeks of culture, three ligament analog tissue constructs were fixed in 4% buffered formaldehyde solution for histological and immunohistoflurorescent examination. They were then dehydrated through alcohol gradients, cleared, and embedded in paraffin blocks. Histological sections (10 μm) were prepared from these specimens using a microtome (Leica Instruments, Bensheim, Germany) and subsequently stained with hematoxylin-eosin (H&E). In addition, the streptavidin-biotin method was used to evaluate the different types of collagen in the regenerated tissue. Histological sections were labeled with primary monoclonal antibodies (anticollagen type I, type III, and tenascin) at a 1 :200 dilution and left overnight at room temperature (ICN Biochemicals, Aurora, OH). Subsequently, fluorescence conjunct antimice secondary antibodies were administered at a 1 :100 dilution for 1 h.

Biomechanical Testing

After 4 weeks of culture, six specimens each from both the ligament analog tissue constructs and the scaffold group were subjected to biomechanical testing. The specimens were mounted on the lnstron 5543 materials testing system (lnstron, Canton, MA) fitted with a 500 N load cell. After the mounting some phosphate-buffered solution was dropped onto the specimen by a syringe to keep it wet. The system has an accuracy of at least 1% of reading up to a maximum of 2 N. At a test speed of 10 mm/min, a tensile load was applied up to 12 N, then unloaded down to 2 N, and then loaded up again to 12 N for a total of five cycles. After five cycles, the specimen was loaded up to failure. The final cycle was checked for steady-state consistency and taken as the representative load displacement curve for the specimen. The cyclic load of 12 N was chosen to keep the loading within the cell-scaffold tissue construct's elastic limit which was decided according to the pilot experiments of two specimens. The test speed of 10 mm/min was chosen on the basis of earlier trial tests conducted on tendon samples, which showed consistency in the loading patterns. The load-versus-elongation curves obtained for each specimen were compared to those reported in previous studies. The tensile stiffness value was calculated from the linear region of the load- elongation curve.

Histology of Ligament Tissue Constructs

After 4 weeks of culture, the composite of cell sheet/PLLA scaffold tissue construct became a tissue-like structure, as can be seen in FIG. 8. The tissues filled up the macropores of the knitted PLLA scaffold. Histology of the longitudinal sections FIG.8(a,b) showed formation of fibrous matrix bundles with a large number of cells aligned in the matrix. The transverse section FIG. 8(c) showed a large number of cells present in connective tissue that filled and wrapped the scaffold.

FIG. 9 shows the results of the immunohistochemical analysis which indicates that the matrix of neoligament tissues in ligament analog tissue constructs was composed primarily of collagen type I and small amount of collagen type III and tenascin.

The histology of bMSC sheet/knitted scaffold tissue construct after 4-week culture is very similar to that of the neotendon treated with bMSC/knitted scaffold tissue construct at 2 weeks after surgery.14 This ligament analog tissue construct is completely composed of the cells and their synthesized matrix. It possesses more tissues as compared to the previously established in vitro ligament analogs which is composite of collagen gel and separated cells. (Altman et al, 2002). The matrix components of the analogs include collagen type I and type III which are the major matrix components of the natural ligament and tenascin, one of the early marker for embryonic ligament. (Altman et al, 2002; Frank et al, 1988 and Amiel et al, 1984).

Biomechanics of Ligament Analogs

All data obtained were expressed as a mean ± standard deviation. Unpaired t- test was employed to assess statistical significance.

The mean tensile stiffness at the elastic region of ligament analog tissue construct group (n = 6) and the scaffold group (n = 6) were 20.6 ± 1.417 N/mm and 27.6 ± 1.449 N/mm, respectively. The mean tensile stiffness of ligament analog tissue construct group was significantly lower than that of scaffold group (p < 0.05). The failure force of the ligament analog tissue construct group and the scaffold group were 46.68 ± 2.29 N and 43.58 ± 2.41 N, respectively. The failure force of the ligament analog tissue construct group was higher than that of scaffold group (p ± 0.05; FIG. 10). However, the failure force of the ligament analog tissue construct was lower than that of original scaffolds (51.45 ± 3.2 N) without significance (p < 0.05). The stiffness of the original scaffold was 28.4 ± 1.812 N/mm which was not higher than that of scaffold group (p > 0.05). The tissue construct of the present invention had an unexpected result: the bMSC sheet made the ligament analog tissue constructs stronger with a higher failure load with less stiffness.

Example 2 - Dermal or skin grafts

Fabrication of bMSCs Sheet

Following the methods described for Example 1 , the bone-marrow-derived adherent cells exhibited fibroblastlike colony-unit formation (CFU-F). The bMSCs proliferated fast and formed cell sheet within 2 weeks after confluence in the presence of ascorbic acid. When the cell sheets were formed, the cell sheets could be detached from culture substratum, thus creating a sheet of cohesive living cells in a collagen matrix of endogenous origin. Once the cell sheet was assembled onto the macroporous knitted PLLA scaffold, the cell sheets adhered to scaffold after several hours of culture as exhibited by the lack of flotation of cell sheet in the medium. The cells within cell sheet were still alive after assembly as indicated by positive CFDA staining under confocal microscopy (FIG. 11). Figure 11 shows the macromorphology (a), transmission image (b), and viable cells as indicated by positive CFDA staining under confocal microscopy of a bMSC sheet after assembling with knitted PLLA scaffold (c). A tissue construct thus prepared may be used for dermal or skin grafts.

Variations under the Present Invention

A person skilled in the art will appreciate that the method of the present invention may be characterized by providing at least one structure and preparing at least one sheet of mesenchymal stem cells and assembling the structure and sheet of cells to form a tissue construct. The cells are then cultured and permitted to grow further on the structure for a period of time until desired characteristics are obtained. The tissue constructs may then be arranged in a desired order for use as a graft mimicking the tissue to be supplemented or replaced. The sheets can be arranged by contacting the sheets such that a sheet substantially covers another sheet below it, for example, by stacking the sheets one on another. Alternatively, the sheets can be arranged wherein a sheet only partially covers another sheet below it.

Should a tubular graft such as a tendon or ligament graft be desired, the constructs may be rolled around a mandrel to form a tube and further cultured for the cells to grow before being used as a graft. In the rolling of the constructs, a person skilled in the art will appreciate that the constructs may be staggered such that when rolled on the mandrel, the edges or seams of the layers do not coincide but are covered by at least one layer above them. This will allow cells to better proliferate over the edges and provides a stronger joining of the seams. While the use of a form such as a mandrel is taught, a person skilled in the art will appreciate that, depending on the graft prepared or fabricated, that a form such as a mandrel may not be necessary to roll the arranged sheets.

Further while the constructs in the examples were rectangular for ease of understanding the invention, the constructs fabricated under the present invention are not restricted to rectangular shapes. The constructs may be cut into triangles and cones may be rolled from them as dictated by the application. Further square or rectangular constructs may be rolled alone their diagonals to form a spindle-like bundle wherein the middle of the rolled sheet is thicker and the ends thinner.

While assembly with at least one sheet of cells was given in the above examples, the invention may also be used to produce grafts wherein structures may also be rolled or twisted to form cords before being wrapped with at least one sheet of cells. Again, sheets of cells may be added at any stage of the process as needed to obtain a desired outcome. While the examples given above teach rolling of the constructs to form a tubular structure, a person skilled in the art will recognize that the structure of the present invention need not be rolled for grafting into muscle walls, or linings of other body parts, to replace or to reinforce these walls, linings or parts.

For use, the graft of the present invention may be supplied as a kit comprising the construct, packaging and information and optionally instructions and/or illustrations pertaining to the use of the construct.

References

Altman GH, Horan RL, Martin I₁ Farhadi J₁ Stark PR, Volloch V, Richmond JC, Vunjak-Novakovic G, Kaplan DL. Cell differentiation by mechanical stress. FASEB J 2002; 16:270 -272.

Amiel D, Frank C, Harwood F, Fronek J, Akeson W. Tendons and ligaments: a morphological and biochemical comparison. J Orthop Res 1984; 1 :257-265.

Auger et al US Patent No. 5,618,718 Production of a contractile smooth muscle.

Colton Ck. (1995) Implantable bioartificial organs. Cell Transplant. 1995. 4: 415-436.

Frank CB, Woo SL-Y, Andriacchi T, Brand R, Oakes B, Dahners L, Dehaven K, Leis J, Sabison P. Normal ligament: structure, function, and composition. In: Woo SLY, Buckwalter J, editors. Injury and repair of musculoskeletal soft tissues. Park Ridge, IL: American Academy of Orthopaedic Surgeons; 1988. p 45-51.

McAllister et al Pub No US 2002/0188349A1 Tissue engineered blood vessels and apparatus for their manufacture

McAllister et al Pub No.US 6,503,273 B1 (2003) Tissue engineered blood vessels and apparatus for their manufacture

Mikos AG, Sarakinos G, Lyman MD, lngber DE, Vacanti JP, and Langer R. (1993) Prevascularization of porous biodegradable polymers. Biotechnology and Bioengineering. 42 (6): (1993) 716-723 SEP 5 Ouyang HW, Goh JCH, Mo XM₁ Teoh SH, and Lee EH. (2002) The efficacy of bone marrow stromal cell-seeded knitted PLGA fiber structure for Achilles tendon repair. Ann N Y Acad Sci. Jun; 961 : 126-9. 2002.

Ouyang HW, Goh JCH, Thambyah A, Teoh SH, Lee EH. (2003) The use of knitted PLGA and MSCs for Achilles tendon repair in rabbit model. Tissue Engineering. VoI 9, No.3, 431-439, 2003.

Ouyang HW, Goh JCH, Thambyah A, Teoh SH, Lee EH. Knitted poly- lactide-co-glycolide scaffold loaded with bone marrow stromal cells in repair and regeneration of rabbit Achilles tendon. Tissue Engineering. VoI 9, No.3, 431-439, 2003.

Goh JCH, Ouyang HW, Chan CK, Teoh SH, Lee EH. Tissue-engineering approach to the repair and regeneration of tendons and ligaments. Tissue Engineering Vol.9 Sup1 , 31-44, 2003.

Ouyang HW, Goh JCH, Lee EH. Use of bone marrow stromal cells for tendon graft-to-bone healing: histological and immunohistochemical studies in a rabbit model. Am J Sport Med 32(2) 321-327, 2004

Ouyang HW, Goh JCH, Mo XM, Teoh SH, AND Lee EH. (2002) Characterization of anterior cruciate ligament cells and bone marrow stromal cells on various biodegradable polymeric films. Mat Sci Eng C-BIO S 20 (1 -2): 63-69 Sp. Iss. SI MAY 31 2002.

Pouliot R, Larouche D, Auger FA, Juhasz J, Xu W, Li H, Germain L. Reconstructed human skin produced in vitro and grafted on athymic mice.Transplantation. 2002 Jun 15;73(11):1751-7. Sittinger M, Bujia J, Rotter N, Minuth WW and Burmester GR (1996), Tissue engineering and autologous transplant formation: practical approaches with resorbable biomaterials and new culture techniques. Biomaterials 17 (1996), pp. 237-242.

Steffens GCM, Yao C, Prevel P, Markowicz M, Schenck P, Noah EM, and Pallua N (2004) Modulation of Angiogenic Potential of Collagen Matrices by Covalent Incorporation of Heparin and Loading with Vascular Endothelial Growth Factor. Tissue Engineering 10, 1502-1509.

Zhang Y, Huang Z-M₁ Xu X, Lim CT, and Ramakrishna S (2004) Preparation of Core-Shell Structured PCL-r-Gelatin Bi-Component Nanofibers by Coaxial Electrospinning. Chemistry of Materials 16, 3406-3409.

Zhang YZ., Ouyang HW, Lim CT, Ramakrishna S. Huang ZM. Electrospinning of Gelatin fibers and Gelatin/PCLcomposite fibrous scaffolds. J Biomedical Material Research 2004; 72B: 156-165.

Zund G, Ye Q, Hoerstrup SP, Schoeberlein A, Schmid AC, Grunenfelder J, Vogt PR and Turina M (1999), Tissue engineering in cardiovascular surgery: MTT, a rapid and reliable quantitative method to assess the optimal human cell seeding on polymeric meshes. Eur J Cardio-thorac Surg 15 (1999), pp. 519- 524.

Claims

1. A tissue construct comprising at least one biocompatible structure and multipotent cells, wherein the multipotent cells are in a sheet form before assembly with the at least one structure.

2. The tissue construct according to claim 1 , wherein the sheet of multipotent cells comprise one or more layers of multipotent cells.

3. The tissue construct according to claims 1 or 2, wherein the multipotent cells comprise mesenchymal stem cells.

4. The tissue construct according to claim 3, wherein the mesenchymal stem cells are adult and/or embryonic stem cells.

5. The tissue construct according to any one of claims 1 to 4, wherein the cells are isolated from autologous and/or allogenic sources.

6. The tissue construct according to any one of claims 1 to 5, wherein the construct further comprises at least one therapeutic agent.

7. The tissue construct according to claim 6, wherein the at least one therapeutic agent is selected from the group consisting of angiogenic growth factors, angiogenic inhibitors, growth factors, thrombin inhibitors, antithrombogenic agents, thrombolytic agents, fibrinolytic agents, vasospasm inhibitors, calcium channel blockers, vasodilators, antihypertensive agents, antimicrobial agents, antibiotics, inhibitors of surface glycoprotein receptors, anti-platelet agents, antimitotics, microtubule inhibitors, anti-secretory agents, actin inhibitors, remodeling inhibitors, antisense nucleotides, anti-metabolites, antiproliferatives, anticancer chemotherapeutic agents, anti-inflammatory steroid or non-steroidal anti-inflammatory agents, immunosuppressive agents, growth hormone antagonists, growth factors, dopamine agonists, radiotherapeutic agents, peptides, proteins, enzymes, extracellular matrix components, inhibitors, free radical scavengers, chelators, antioxidants, anti-polymerases, antiviral agents, photodynamic therapy agents, and gene therapy agents.

8. The tissue construct according to any one of claims 1 to 7, wherein the at least one structure is in the form of a sponge, foam, mesh of fibers, sheet, woven or knitted material.

9. The tissue construct according to any one of claims 1 to 8, wherein the at least one biocompatible structure comprises at least one natural and/or synthetic material.

10. The tissue construct according to claim 9, wherein the at least one synthetic material is selected from the group consisting of ceramic material, biosorbable and biodegradable material.

11. The tissue construct according to claim 10, wherein the biosorbable material is selected from the group consisting of poly (L-lactide) (PLLA), polyglycolide (PGA), polylactide (PLA), polycaprolactone (PCL), poly(glaxanone), poly(orthoesters), poly(pyrolicacid), poly(phosphazenes and their derivatives.

12. The tissue construct according to claim 9, wherein the at least one natural material is selected from the group consisting of collagen, gelatin, silkworm silk, spider silk, allogenic fascia tissue, autologous fascia tissues, and alimentary canal/intestinal submucosa.

13. The tissue construct according to any one of claims 1 to 12, wherein the construct is suitable for use as a graft.

14. The tissue construct according to any one of claims 1 to 13, wherein the construct is suitable for use as a graft for connective tissue.

15. The tissue construct according to any one of claims 1 to 14, wherein the construct is suitable for use as a graft for tendon and/or ligament.

16. The tissue construct according to any one of claims 1 to 13, wherein the construct is suitable for use as a graft for skin.

17. A kit comprising the tissue construct according to any one of claims 1 to 16.

18. A method of preparing a tissue construct, the method comprising: providing at least one biocompatible structure; preparing at least one sheet of multipotent cells, and assembling the at least one structure and at least one sheet of multipotent cells to obtain the tissue construct.

19. The method according to claim 18, wherein the sheet of multipotent cells comprises one or more layers of multipotent cells.

20. The method according to claim 18 or 19, wherein the assembling further comprises stacking, rolling, folding and/or wrapping the tissue construct.

21. The method according to any one of claims 18 to 20, wherein the tissue construct is further cultured after assembly.

22. The method according to any one of claims 18 to 21 , wherein the multipotent cells comprise mesenchymal stem cells.

23. The method according to claim 22, wherein the mesenchymal stem cells are adult and/or embryonic stem cells.

24. The method according to any one of claims 18 to 23, wherein the cells are isolated from autologous and/or allogenic sources.

25. The method according to any one of claims 18 to 24, the method further comprising adding at least one therapeutic agent to the construct.

26. The method according to claim 25, wherein the at least one therapeutic agent is selected from the group consisting of angiogenic growth factors, angiogenic inhibitors, growth factors, thrombin inhibitors, antithrombogenic agents, thrombolytic agents, fibrinolytic agents, vasospasm inhibitors, calcium channel blockers, vasodilators, antihypertensive agents, antimicrobial agents, antibiotics, inhibitors of surface glycoprotein receptors, anti-platelet agents, antimitotics, microtubule inhibitors, anti-secretory agents, actin inhibitors, remodeling inhibitors, antisense nucleotides, anti-metabolites, antiproliferatives, anticancer chemotherapeutic agents, anti-inflammatory steroid or nonsteroidal anti-inflammatory agents, immunosuppressive agents, growth hormone antagonists, growth factors, dopamine agonists, radiotherapeutic agents, peptides, proteins, enzymes, extracellular matrix components, inhibitors, free radical scavengers, chelators, antioxidants, anti-polymerases, antiviral agents, photodynamic therapy agents, and gene therapy agents.

27. The method according to claims any one of claims 18 to 26, wherein the at least one structure is in the form of a sponge, foam, mesh of fibers, sheet, woven or knitted material.

28. The method according to any one of claims 18 to 27, wherein the at least one biocompatible structure comprises at least one natural and/or synthetic material.

29. The method according to claim 28, wherein the at least one synthetic material is selected from the group consisting of ceramic material, biosorbable and biodegradable material.

30. The method according to claim 29, wherein the biosorbable material is selected from the group consisting of poly (L-lactide) (PLLA), polyglycoiide (PGA), polylactide (PLA), polycaprolactone (PCL), poly(glaxanone), poly(orthoesters), poly(pyrolicacid), poly(phosphazenes and their derivatives.

31. The method according to claim 28, wherein the at least one natural material is selected from the group comprising collagen, gelatin, silkworm silk, spider silk, allogenicfascia tissues, autologous fascia tissues, and alimentary canal/ intestinal submucosa.

32. The method according to any one of claims 18 to 31 , wherein the construct is suitable for use as a graft.

33. The method according to any one of claims 18 to 32, wherein the construct is suitable for use as a graft for connective tissue.

34. The tissue construct according to any one of claims 18 to 33, wherein the construct is suitable for use as a graft for tendon and/or ligament.

35. The tissue construct according to any one of claims 18 to 32, wherein the construct is suitable for use as a graft for skin.