HK1180253A - Methods for complex tissue engineering - Google Patents

Description

Method for complex tissue engineering

Cross Reference to Related Applications

This application claims benefit of U.S. provisional application No. 61/354,869, filed on 6/15/2010, which is herein incorporated by reference in its entirety.

Technical Field

The present invention relates generally to bioengineered tissues. In particular, it relates to a method of producing a tissue graft having more than one tissue component.

Background

Tissue dysfunction may be caused by trauma, genetic or surgical causes. For mild lesions, some tissues can regenerate themselves, while others, especially those of a hypo-blood supplying nature, are hardly regenerated. Many attempts have been made to provide treatment options for tissue dysfunction, including growth factor therapy, cell therapy, and gene therapy. However, these attempts are only useful when the degree of tissue dysfunction is not significant. When tissue dysfunction is large, replacement therapy by surgical methods is necessary. In this regard, tissue engineering methods for growing and culturing three-dimensional (3D) tissue-like structures composed of cells and biomaterial-based scaffolds, supplemented with growth-stimulating signals, offer great promise for tissue replacement therapy.

In the last two decades, great progress has been made in biomaterials and manufacturing technologies, stem cell technologies and bioreactor technologies, making it easier to prepare 3D tissue-like structures with similarities to native tissue structure and function for replacement purposes. However, all organs are composed of complex tissues with more than one tissue component with unique structure, cell type and function. A complex tissue is a tissue or organ having more than one tissue component. On the other hand, most, if not all, functional tissues are composed of more than one component and have unique structures and cell types. Examples of complex organizations include: osteochondral grafts consisting of bone and cartilage with organized calcified cartilage regions; a spinal motion segment consisting of a pair of bone pieces connected by a pair of thin cartilaginous endplates and an intervertebral disc sandwiched therebetween having an annulus fibrosus encapsulating a nucleus pulposus; ligamentous bone graft consisting of two pieces of bone with a ligament strip attached in between. The inherent heterogeneity of complex tissues makes the engineering of complex tissues or the engineering of organs composed of complex tissues a challenge in the art.

Bioengineering complex tissues is extremely challenging because: (1) the need for multiple cell types for different tissue components complicates the source issue; (2) in view of the critical functions that tissue junctions (tissue interfaces) play in ensuring the normal function of complex tissues, a variety of biological and stable tissue junctions are required; (3) the need to maintain multiple phenotypes and functions complicates culture conditions; and (4) many irregularities in the morphological, structural and functional properties of different tissue components and their linkers exist in complex tissues, but are difficult to mimic.

It is almost clinically impossible to provide multiple parenchymal cells for different tissue components of complex tissues. This is because multiple biopsy samples of different tissue components from healthy tissue counterparts are too invasive and cells in dysfunctional tissue are often abnormal. Moreover, mature cells have a limited lifespan and proliferative potential. Pluripotent stem cells capable of differentiating to all phenotypes of different tissue components of a complex tissue being engineered provide a solution to this problem. However, most of the current attempts to bioengineer complex tissues involve the use of one or more sources of cells, making clinical application difficult, or a combination of stem and mature cells isolated from different sources, which is a very complex solution.

Biological and functional tissue junctions, such as osteochondral junctions, are important features of complex tissues. Only recently, the importance of joint tissue engineering has begun to receive increasing attention (Broom, et al, J Anat., 135 (65-82 (1982); Yang et al, Tissue Eng Part B Rev. 2009; 15(2):127-41 (2009) keeney, et al, Tissue Eng Part B Rev., 15(1):55-73(2009)). Previous methods include separately fabricating scaffolds for bone and cartilage portions prior to assembly using solid-free-form fabrication techniques to construct scaffolds with heterogeneity (e.g., gradient porosity), and directly encapsulating cells in a hydrogel prior to assembly using photopolymerization (Sherwood, et al,Biomaterials23(24) 4739-51 (2002), Alhadlaq, et al,J. Bone Joint Surg Am., 87(5):936-44 (2005)). However, the results far fall short of the ultimate goal of mimicking the compartmentalized organization of the structure and achieving functional mechanical properties of the native osteochondral junction, with many challenges to be solved (Broom, et al,J Anat135(1) 65-82 (1982), Yang, et al,Tissue Eng Part B Rev.,15(2) 127-41(2009), and Keeney, et al,Tissue Eng Part B Rev., 15(1):55-73 (2009))。

first, there is still a need to define the optimal combination of cells and scaffold. Bioceramics, such as hydroxyapatite, are the most popular choice for bone parts (osteop-part) or bones (bone), while synthetic polymers, such as poly-lactic-co-glycolic acid (PLGA), are commonly used for cartilage parts (chondro-part) or cartilage (carage) (Kreklau, et al,Biomaterials20(18) 1743-9 (1999), Gao, et al,Clin Orthop Relat Res427(Suppl): S62-6 (2004), Huang, et al, biomaterials.2007; 28(20):3091-,J Orthop Res., 25(10):1277-90 (2007). Chondrocytes isolated from different regions behave differently, with the potential to engineer cartilage tissue with biomimetic compartmentalized tissue (Klein, et al,Osteoarthritis Cartilage, 11(8):595-602 (2003). For example, chondrocytes from the deep regions of articular cartilage have been cultured in calcium phosphate, which act as scaffolds for bone parts, in the absence of a corresponding scaffold for cartilage parts, these cells leading to the formation of calcified regions in vitro (Yu, et al,Biomaterials18:1425 (1997), Allan, et al,Tissue Eng13:167-,Biomaterials, 27(22):4120-31 (2006)). Clinical availability of chondrocytes will likely be problematic due to (i) the need to harvest biopsy samples from non-weight bearing areas, and (ii) the limited proliferative potential of these cells in vitro.

Multipotent and pluripotent stem cells (pluripotent stem cells), such as bone marrow-derived Mesenchymal Stem Cells (MSCs), are promising because of their self-renewal capacity and multiple differentiation potential (Pittenger, et al,Science284(5411) 143-7 (1999), Pittenger, et al,Circ Res95(1) 9-20 (2004), and Le Blanc, et al,Lancet, 363(9419):1439-41 (2004)). Although MSCs hold promise, in using MSCs and silk scaffolds (Augst, et al,J R Soc Interface929-39, (2008) or MSC and poly D, L-lactic acid scaffold (Tuli, et al,Tissue Eng10: 1169-. In Tuli studies, although the complex was cultured in a medium capable of simultaneously maintaining cartilage formation and osteogenic phenotype, the culture conditions were unable to support the formation of calcified cartilage junctions (Tuli, et al, Tissue Eng, 10:1169-1179 (2004))。

second, integration with host cartilage is a common problem with most existing strategies, as multiple cylindrical plugs (plugs) are typically used to fill defects. These plugs are often irregularly shaped, resulting in limited integration and increased contact pressure due to non-uniformities in the articular surface (Yang, et al, Tiss)ue Eng Part B Rev15(2) 127-41(2009), Keeney, et al,Tissue Eng Part B Rev15, (1) 55-73, (2009), Martin, et al,J. Biomech. 40(4):750-65 (2007)). Cell layer technology, which involves laminating multiple confluent cell monolayers, appears to be a flexible and scalable technology capable of preparing tissues with heterogeneity (Shimizu, et al,Biomaterials, 24(13):2309-16 (2003)). However, multiple lamination requires time because it is time consuming to adhere one layer to the previous layer. Thus, the time for an open surgery is very long. Therefore, only thin-layer tissues having high cellularity such as epithelial tissues and endothelial tissues can be prepared using this method. Tissues with a large matrix, tissues requiring mesenchymal cell types, tissues with a load-bearing function and with irregular tissue junctions, etc. cannot be prepared using this method.

Thus, bioengineering complex tissues with heterogeneity and irregularity requires more flexible and scalable technology that allows for the formation and maintenance of multiple tissue types with multiple stable tissue junctions from simple cell and material sources, with better therapeutic efficacy and lower cost.

It is therefore an object of the present invention to provide a flexible and scalable method of generating complex organizations composed of more than one organization components.

Another object of the present invention is a method of repairing a complex tissue defect by implanting the complex tissue scaffold disclosed herein at a site where complex tissue replacement is desired.

Summary of The Invention

The present invention describes a simple, highly flexible and scalable platform for preparing functional complex organizations with heterogeneity and irregularity. A representative method of using this platform involves combining pluripotent or multipotent cells such as stem cells with biomaterials to make a variety of undifferentiated or naive (nave) subunits. In some embodiments, these undifferentiated or naive (nameive) subunits are microencapsulated pluripotent or multipotent cells. The method further involves exposing at least portions of the undifferentiated or naive (nameive) subunits to different environments for inducing differentiation to the different lineages required for the complex tissue, and combining then functional subunits with or without undifferentiated or naive (nameive) subunits to form a bioengineered complex tissue that mimics native complex tissue in structural irregularities and heterogeneity. In some embodiments, undifferentiated or naive (nave) subunit differentiation is further induced by exposing undifferentiated pluripotent or multipotent cells to suitable microenvironments (including but not limited to differentiation media and mechanical loads) capable of maintaining multiple phenotypes in bioengineered complex tissues. In other embodiments, undifferentiated pluripotent or multipotent cells are induced to differentiate by interaction with various differentiated progeny of the stem cells.

Also provided are methods of making a bioengineered tissue graft involving combining two or more functional subunits, optionally separated by undifferentiated encapsulated pluripotent or multipotent cells, wherein each of the two or more functional subunits are encapsulated pluripotent or multipotent cells that can be induced to differentiate into different cell types; and culturing the combined functional units to form a bioengineered tissue graft that mimics the structural and functional characteristics of the complex tissue.

Suitable pluripotent or multipotent cells for use in cell transplantation include induced pluripotent stem cells, embryonic stem cells, fetal stem cells, cord blood stem cells, bone marrow-derived stem cells, and adipose tissue-derived stem cells.

Pluripotent or multipotent cells are encapsulated in a biomaterial barrier such as an extracellular matrix biomaterial. In a preferred embodiment, the extracellular matrix biomaterial is collagen. The cells may be encapsulated into any suitable structure, such as microspheres, cubes, rings, or micro-rods.

In some embodiments, the encapsulated pluripotent or multipotent cells are induced to differentiate using any available means, such as chemical induction, genetic manipulation, or by reconstituting the biological and mechanical microenvironment.

In some embodiments, the method further involves fine-tuning the structural and functional properties of the bioengineered tissue graft by exposing the bioengineered tissue graft to suitable biological, chemical and physical co-culture conditions. In some embodiments, the combined functional units are cultured in the presence of conditioned media, growth factors, cytokines, serum, and combinations thereof. In some embodiments, the combined functional units are cultured under chemical conditions suitable to promote functional and structural features of complex tissues. Chemical conditions include antioxidants, acids, bases, oxygen tension, and combinations thereof. In some embodiments, the functional unit of the combination is cultured in the presence of a force selected from the group consisting of torsion, compression, tension, and combinations thereof.

Complex tissues associated with the disclosed methods include osteochondral tissue, intervertebral discs, spinal motion segments; ligamentous bone tissue, cardiac strips with myocardial muscle containing a blood-supplying network, a highly organized layered growth plate with cartilage capable of lengthening and thickening bone tissue, and a liver patch with fully blood-supplied hepatocytes. In a preferred embodiment, the complex tissue is osteochondral tissue with zoned tissue. In other embodiments, the complex tissue is a spinal motion segment having a multi-lamellar structure of the annulus fibrosus.

Another embodiment provides a method of repairing a tissue defect involving the use of the disclosed bioengineered tissue graft at a site of tissue damage in a subject. Another embodiment provides a method of repairing a tissue defect involving transplanting a combination of naive (meive) and/or differentiated subunits at a site in need thereof.

In another embodiment, the combined naive (nave) and differentiated subunits may be cultured, prior to transplantation, under biological, chemical and/or physical culture conditions suitable for fine-tuning the structural and functional properties of the bioengineered complex tissue. Suitable biological culture conditions include customized media, conditioned media, growth factors and cytokines, serum and other blood products, and combinations thereof. Suitable chemical conditions include antioxidants, acids, bases, oxygen tension, and combinations thereof. Suitable physical conditions include mechanical loading of a force selected from the group consisting of torsional, compressive, tensile, and combinations thereof.

Also provided are methods of producing a calcified region junction in a bioengineered tissue construct, the method involving culturing an in vitro construct having first, second and third layers to induce a calcified region between the first and third layers; wherein the first layer comprises microencapsulated pluripotent or multipotent cells that can be treated to induce osteogenic differentiation; wherein the second layer is adjacent to the first layer, separates the first and third layers, and comprises undifferentiated microencapsulated pluripotent or multipotent cells; wherein the third layer is adjacent to the second layer and comprises pluripotent or multipotent cells that can be treated to induce chondrogenic differentiation.

Also provided are methods of producing complex tissue constructs involving culturing two layers of encapsulated stem cells that can be induced to differentiate by culturing the two layers of encapsulated stem cells under conditions that induce formation of the complex tissue construct, the two layers of encapsulated stem cells being linked together by a layer of undifferentiated encapsulated stem cells.

Also provided are bioengineered tissue grafts produced by the disclosed methods.

Also provided is a bioengineered osteochondral tissue construct comprising a first, second and third layer, wherein the first layer comprises microencapsulated pluripotent or multipotent cells that can be treated to induce osteogenic differentiation, wherein the second layer is adjacent to the first layer, separates the first and third layers, and comprises undifferentiated microencapsulated multipotent or multipotent cells; wherein the third layer is adjacent to the second layer and comprises pluripotent or multipotent cells that can be treated to induce chondrogenic differentiation. In a preferred embodiment, the bioengineered construct has a calcified regional junction between the first and third layers.

Also provided are methods of treating osteochondral injury in a subject involving administering the disclosed bioengineered osteochondral constructs to the subject at the site of the osteochondral injury.

Also provided are methods of producing a bioengineered spinal motion segment, the methods involving inducing microencapsulated pluripotent or multipotent stem cells to form osteogenic subunits; combining the osteogenic subunits with microencapsulated pluripotent or multipotent stem cells that can be induced to form chondrogenic subunits, wherein the combination of osteogenic subunits and chondrogenic subunits form bone blocks comprising endplates; the intervertebral disc is sandwiched with an annulus fibrosus encapsulating the nucleus pulposus and a bone block comprising endplates to form a spinal motion segment. The intervertebral discs may be natural, bioengineered, or synthetic.

Brief description of the drawings

FIG. 1 is a flow chart summarizing a method of complex tissue formation.

Fig. 2 is a flow chart showing a method of preparing an osteochondral graft of bone and cartilage having calcified cartilage joints as an example of complex tissue engineering.

Fig. 3A is a linear plot (n =4) showing the relative amounts (μ g) of glycosaminoglycans (GAGs) (♦) and Hydroxyproline (HYP) (■) in microspheres cultured in chondrogenic medium as a function of time (0, 14, and 21 days). Fig. 3B is a linear plot (n =4) showing the GAGs/HYP ratio of chondrogenic microspheres (♦) compared to normal rabbit knee cartilage (■) as a function of time (0, 14, and 21 days). Fig. 3C is a linear graph (n =5) showing the percentage of calcium/dry weight (w/w%) in the osteogenic microspheres as a function of time (0, 14 and 21 days).

Fig. 4 is a bar graph showing the cumulative amount (pg) of BMP2 secreted from osteogenically differentiated MSC-collagen microspheres as a function of culture time (7, 14 and 21 days).

Fig. 5 is a bar graph of tensile strength (kPa) of the osteochondral constructs bioengineered 7 days (solid bars) and 21 days (open bars) after the following conditions: 1) without compression, with chondrogenic medium ("XC"); 2) without compression, with normal growth medium ("XN"); 3) with compression using chondrogenic medium ("CC"), and 4) with compression using normal growth medium ("CN"). P = 0.05.

Detailed description of the invention

I. Definition of

As used herein, "complex tissue" refers to a collection of two or more cell types that perform separate biological functions. Exemplary complex tissues include, but are not limited to, osteochondral tissue and organ tissue.

As used herein, "bioengineered complex tissue" and "bioengineered tissue grafts" refer to compositions or structures that, when transplanted into complex tissue, produce tissue that mimics the structure and function of the complex tissue.

As used herein, a "undifferentiated or naive (na-mei) subunit" is an undifferentiated cell biomaterial subunit prepared by techniques such as microencapsulation.

As used herein, a "functional subunit" is a differentiated tissue micro-substance (micromassases) that requires the function of native tissue.

As used herein, "encapsulated in a microsphere" refers to a nanofiber microsphere having cells embedded therein as a result of a phase transition of the material forming the microsphere.

As used herein, "stem cell" generally refers to an undifferentiated cell, regardless of source, including multipotent cells, pluripotent cells, dedifferentiated cells, embryonic stem cells, bone marrow mesenchymal stem cells, and induced pluripotent stem cells. The stem cells may be embryonic or adult stem cells.

As used herein, "totipotency" refers to the ability of a single cell to divide and produce all differentiated cells in an organism, including extraembryonic tissues.

As used herein, "pluripotency" refers to the ability of a single cell to differentiate into cells of any of the three germ layers (endoderm (e.g., internal gastric mucosa, gastrointestinal tract, lung), mesoderm (e.g., muscle, bone, blood, urogenital system), or ectoderm (e.g., epidermal tissue and nervous system)). Pluripotent cells cannot develop into a fetal or adult animal because they lack the ability to contribute to extraembryonic tissues such as the placenta.

As used herein, "multipotent" refers to cells that have the ability to differentiate into cells of multiple cell lines, but not all three germ layers.

As used herein, "subject" refers to any individual who is the target of administration. The subject can be a vertebrate, e.g., a mammal. Thus, the subject may be a human. The term does not indicate a particular age or gender. A patient refers to a subject afflicted with a disease or condition. The term "patient" includes human and veterinary subjects.

Method for forming complex tissue

Bioengineering complex tissues requires more flexible and scalable technology that allows for the formation and maintenance of multiple tissue types with multiple stable tissue junctions from simple cell and material sources. The disclosed method uses the concept that the tissue is composed of functional subunits that are of small volume, e.g., 10^-6ml and with, for example, 100-. The disclosed methods also use the multiple differentiation potentials of various types of stem cells and the interactions between undifferentiated stem cells, differentiating stem cells and their differentiated progeny. The disclosed methods further use the ability of cells to engineer their existing matrices (including but not limited to extracellular matrices and microenvironments) for fine-tuned differentiation of cells into desired tissue structures. The engineered matrix is composed of cells, preferably of cellsStem cells with various signal responses are formed. In particular, the present disclosure provides methods for providing conditions for bioengineered complex tissues in vitro by mimicking or reconstructing the biological and mechanical microenvironment of the tissue as much as possible such that a variety of cellular or tissue phenotypes can be maintained and functional complex tissues can be grown. The method integrates multiple functional subunits of different tissue components of complex tissues to bioengineer the complex tissues. The examples show that the disclosed method is effective at 5 weeks in preparing osteochondral grafts with calcified cartilage junctions from a single stem cell source and a single biomaterial (figure 2). Its efficacy and simplicity make complex tissue engineering possible and affordable.

A. Cells

Representative cells useful in the disclosed methods include, but are not limited to, pluripotent or multipotent cells, such as adult or embryonic stem cells, as the sole source of cells for preparing various tissue components of complex tissues. The stem cell may be an Induced Pluripotent Stem Cell (iPSC), an embryonic stem cell, a cord blood stem cell, a bone marrow-derived stem cell such as a Mesenchymal Stem Cell (MSC), an adipose tissue-derived stem cell such as a mesenchymal stem cell, or the like obtained by a viral or non-viral method. A clinically viable stem cell source such as bone marrow-derived mesenchymal stem cells is preferred because it is readily available, and is ethically and socially more acceptable.

The cells may also be commercially available stem cells. They may be obtained from companies selling stem cells or any source approved by regulatory agencies. For example, they may be purchased from Beike Biotechnology.

B. Extracellular matrix (ECM) biomaterials

The composition includes at least one biological material that must be capable of providing support to the cells, interacting with the cells to allow cell growth without introducing toxicity, and allowing cell migration and infiltration. The biomaterial may be different types of collagen such as I, II and type III, or well-supported cellsAny material that grows and migrates and has phase transition properties under conditions that are mild enough to support cell survival, such as fibrin and hyaluronic acid. The collagen used may be of bovine origin, such as the American Food and Drug Administration (FDA) approved skin equivalent Integra^RTMAnd Apligraf^RTMAnd soft tissue fillers or products such as DermaLive and DermaDeep (Bergeret-Galley, et al),Aesthetic Plast.Surg249-55 (2001), or for the treatment of urinary incontinence (Corcos, et al,Urology65(5) 898-904 (2005)). The biological material may be derived from natural or synthetic sources, which may be induced to reconstitute into a solid form under specific conditions sufficiently mild to support cell survival and growth. The biological material may be produced from isolation or extraction from various animal sources, such as rat tail, pig skin, bovine achilles tendon, or human placenta. Preferably, the biological material is separated from different fractions during extraction, such as acid-soluble, pepsin-soluble or insoluble fractions.

The composition may further comprise a second biomaterial which may be a proteoglycan or glycosaminoglycan ("GAG") obtained from shark cartilage, fibrin, elastin or hyaluronic acid. The first biomaterial may interact with living cells or with the second biomaterial in a manner such that the interaction results in cellular reactions in growth and differentiation and changes in the physical properties of the microspheres, such as volume of structure, biomaterial density, cell density, mechanical properties, stability, and the like.

Suitable biomaterials also include hydrogels such as alginate gels gelled by the addition of calcium, which are made under conditions sufficiently mild to maintain high cell viability after encapsulation without the use of organic solvents or other substances toxic to cells and without harsh conditions.

The biological material does not affect the stem cell properties of the multipotent or pluripotent cells. It provides an optimal scaffold that allows the engineering of biomaterials into natural tissue-like matrices by the differentiated cells and supports the growth of new tissue components or tissue junctions during complex tissue engineering processes. The biological material may be synthetic, natural, or a combination thereof. Preferred biomaterials include, but are not limited to, biomaterials that are capable of self-assembling into a fibrous network that captures the stem cells prior to their differentiation into cells of different progeny or lineages. Biomaterials include, but are not limited to, collagen, fibrinogen, elastin, self-assembling peptides, and combinations thereof.

C. Complex tissue formation

Undifferentiated cells, e.g., stem cells, are combined with biological material to form complex tissues. In one embodiment, microencapsulation is used to convert the cell/biomaterial combination into a number of undifferentiated or naive (meive) subunits. Microencapsulation does not affect the stem cell properties of undifferentiated cells. Preferred methods of microencapsulation include, but are not limited to, those described in U.S. published application No. 2008/0031858 to Chan et al, or Chan et al,Biomaterials28: 4652-4666 (2007), both of which are incorporated by reference in their entirety where permissible. Microencapsulation allows undifferentiated cells to be captured in biomaterial structures of controlled size in the form of microspheres, cubes or rods, without affecting the properties of the stem cells, including their self-renewal capacity and multi-differentiation potentialChan, Etc. of ., Biomaterials,28:4652-4666 (2007)). The structures or subunits of these micron-sized biomaterials can be used for subsequent differentiation or assembly to form complex tissues.

The naive (na-meive) subunits are differentiated into different lines or progeny corresponding to different tissue components in complex tissues using methods known in the art, including, but not limited to, chemical induction, remodeling of biological microenvironments, and genetic manipulation. The thus treated naive (meive) subunit becomes a functional subunit, i.e. a differentiated tissue microsome having the biological function of native tissue. Examples include cartilage-like microsubstances prepared by chondrogenic differentiation of naive (na-meive) MSC-collagen microspheres, and bone-like microsubstances prepared by osteogenic differentiation of native MSC-collagen microspheres (table 1).

Table 1 mean and Standard Deviation (SD) of the relative compositions for calcium and phosphorus in the different groups.

The functional subunits comprise differentiated progeny of the engineered scaffold and stem cells. For example, the compositions can be prepared using Hue, et al,Biomaterials29:3201-3212 (2008) for the formation of chondro-like functional subunits from naive (na-meive) subunits, Chan et al,Tissue Eng Part C Methods16(2) 225-35 (2010) the method described in the claims forms a bone-like functional subunit from a naive (meive) subunit. Following differentiation of the functional subunits, the functional subunits are combined to form complex tissue-like structures. In some embodiments, functional subunits from the same cell type are combined and then combined with functional subunits from another cell type. In one embodiment, the subunits are combined in a random fashion by mixing different subunits in suspension, and then co-culturing the subunits at a high subunit density. In another embodiment, the subunits are combined in a predetermined pattern by integrating the subunits in an appropriate configuration that mimics the heterogeneity and irregularity of native tissue. This can be done in a suitable configuration at a suitable point in time. The duration of the combination should also be optimized according to different complex organizations. Different complex tissues have different designs, and the base line is a structure that mimics natural tissue as much as possible.

In other embodiments, the disclosed methods utilize direct or indirect interactions between differentiated progeny and undifferentiated stem cells of stem cells in appropriate configurations and conditions such that the differentiated progeny also induce differentiation of the undifferentiated stem cells to lineages that are difficult to simply induce by chemical induction. Differentiated progeny of stem cells are obtained from undifferentiated stem cell sources by a number of different methods at early or late stages of the differentiation pathwayThe resulting differentiated cells, the methods include known chemical induction protocols, known genetic manipulation protocols and indirect induction methods when the differentiation protocol is unknown. If the differentiation protocol of chondrocytes is unknown, for example, an indirect method is to capture mature cells from readily available animal or human sources in a biomaterial by microencapsulation and allow the mature chondrocytes to grow and remodel the biomaterial scaffold into a biomaterial scaffold that mimics the natural tissue microenvironment, then discard the mature cells and replant the subunits with undifferentiated stem cells. The matrix and biological environment reconstituted from the mature cells induces the reimplanted undifferentiated stem cells into chondrocyte-like cells that are differentiated progeny of the undifferentiated stem cells (Cheng, et al,Tissue Engineering Part C. 15(4):697-706 (2009))。

the interaction between differentiated progeny of stem cells and their undifferentiated counterparts includes co-culturing functional subunits having differentiated progeny of stem cells with naive (na-meive) subunits made from undifferentiated stem cells under suitable culture conditions having a suitable 3D configuration for a suitable period of time. This interaction may be direct contact between differentiated progeny and undifferentiated stem cells, or indirect induction by factors secreted from differentiated progeny. The interaction also involves secreted factors from the differentiated stem cells. This interaction can be used to induce undifferentiated stem cells into second or third or nth lineages or progeny that cannot be directly differentiated due to unknown chemical differentiation protocols. For example, it is difficult to directly differentiate stem cells to form uncalcified cartilage and calcified chondrocytes (hypertrophic chondrocytes) with appropriate compartmentalized tissue at the same time. It is possible to induce such a differentiated tissue by appropriately configuring the undifferentiated or native subunits with functional subunits made from differentiated progeny (in this case osteogenic and chondrogenic). The undifferentiated stem cells were induced to differentiate into the hypertrophic lineage that constituted a calcified cartilage layer, possibly due to factors secreted by differentiated progeny in functional subunits, including but not limited to BMP 2.

The differentiated subunits of the above combinations are used to replace dysfunctional complex tissues. Alternatively, the differentiated subunits thus combined may be cultured for an appropriate period of time under appropriate conditions for fine-tuning the structural and functional properties of the tissue. A schematic showing the process disclosed herein is provided as in fig. 1.

Cells can be differentiated by altering or modifying biological, chemical, physical culture conditions, or a combination thereof. Biological conditions can be modified by including conditioned media from other cells, co-culturing with other cells, including growth factors and cytokines, serum and other blood products, and combinations thereof. Chemical conditions may be modified by including antioxidants, acids, bases, or modifying oxygen tension, among others. The physical conditions may be modified by varying the mechanical loading of different modes of force, such as torsional, compressive and tensile, or any combination of these, at different degrees and frequencies, etc. Bioreactors may be used, in which the biological, chemical and mechanical microenvironment of cells may be tailored and optimized for the production of different complex tissues.

Complex organization

Examples of complex tissues that may be formed according to the methods described herein include osteochondral grafts, which consist of bone and cartilage with organized calcified cartilage regions; a spinal motion segment consisting of a pair of bone pieces connected by a pair of thin cartilaginous endplates and an intervertebral disc sandwiched therebetween having an annulus fibrosus encapsulating a nucleus pulposus; a ligamentous bone graft consisting of two pieces of bone, said pieces of bone having a ligament strip attached in between; a cardiac strip of myocardial muscle with a network of blood supplies; a highly organized layered growth plate with cartilage capable of lengthening and thickening bone tissue and a liver patch with fully blood-supplied hepatocytes. A schematic diagram for forming an osteochondral graft, for example, as shown in figure 2.

Using the spinal motion segment as an example, naive (naive) microspheres are exposed to osteogenic differentiation conditions to form osteogenic subunits, which are then combined to form a bone mass. The natural subunits are exposed to chondrogenic differentiation conditions to produce cartilage subunits, which are then combined to form thin and large layers that act as endplates. Osteochondral junctions between bone pieces and endplates may be formed using the same methods as shown in the osteochondral graft example-constructs in three layers are formed with undifferentiated cells (e.g., MSCs) in the middle layer and co-cultured in an optimal co-culture medium, such as chondrogenic in this case. Two such osteochondral units are used to form the spinal motion segment, with the intermediate disc portions being recombined by multi-layer co-culture. When the differentiation protocol is unknown, the discotic portion can be obtained by the above-described method. Before the multilayer construct is ready for transplantation, the multilayer construct can be loaded onto a bioreactor that provides mechanical loading that mimics natural tissue for better integration of different tissues and continued tissue growth.

The invention will be further understood by reference to the following non-limiting examples.

The invention will be further understood by reference to the following non-limiting examples.

Examples

Example 1: bone marrow puncture and rabbit mesenchymal stem cell (rMSC) isolation

Three month old New Zealand white rabbits, with an average weight of 3.5kg, were anesthetized by intramuscular injection of a mixture of 10% ketamine hydrochloride (0.35ml/kg) and 2% xylazine (0.25 ml/kg). About 5ml of bone marrow was aspirated from the tibia. Following Ficoll-Hypague gradient separation, monocytes at the interface were collected and cultured in Dulbecco's Modified Eagle Medium (DMEM) containing 10% Fetal Bovine Serum (FBS) and antibiotics. The medium was renewed 10 days after inoculation and then refilled every 2 days thereafter. About 5-7 days after the initial plating, visible colonies of adherent cells were found. After reaching confluence (about 12-14 days after initial plating), cells were detached by 0.25% trypsin/EDTA for sub-culture.

Alternatively, rMSCs may be purchased from Beijing YiKeLiHao Biotechnology Co., Ltd.

Example 2: culture of rMSC

In Dulbecco's modified Eagle's Medium (DMEM-HG) with high glucose, 10% Fetal Bovine Serum (FBS), 100U/ml penicillin, 100mg/ml streptomycin, 1.875mg/ml sodium bicarbonate (NaHCO)₃) rMSC were cultured in complete medium consisting of 0.02M HEPES and 0.29mg/ml L-glutamine. The final pH of the medium was adjusted to 7.4 with 1N sodium hydroxide (NaOH). Live cells in the medium were separated from dead cells after 24 hours by adhesion selection, i.e., the cells were cultured for 24 hours, and then the adhered cells were separated from dead cells in the medium. Cells were maintained in complete medium and refilled every 3 days. Sub-confluent (subonfluence) rMSCs were separated by 0.25% trypsin/EDTA. Cells from passage 2-3 were used for the subsequent microencapsulation step.

Example 3: preparation of juvenile (meive) subunit-collagen-rMSC microspheres

The ice-cold rat tail type I collagen (Becton Dickenson) was neutralized with 1N NaOH and further diluted with complete medium to a final concentration of 2 mg/ml. Aliquots of rMSCs of P2-P3 in complete medium were rapidly mixed with neutralized collagen solution in an ice bath, resulting in a cell-matrix mixture with a final cell density of 1250 cells/2.5. mu.l drops. The droplets were dispensed into 35mm diameter Petri dishes (Sterlin) and the bottom layer was covered with a UV-irradiated sealing film. In the presence of 5% CO₂After incubation at 37 ℃ for 1 hour in humid air, the droplets gelled to form solid rMSC-collagen microspheres, which were then gently rinsed into Petri dishes using complete medium and cultured for 3 days before the differentiation step.

Example 4: formation of cartilage-like functional subunits

Chondrogenic differentiation of the rMSC-collagen microspheres was induced by culturing the rMSC-collagen microspheres as a suspension in a chondrogenic differentiation inducing medium 3 days after formation of the naive (naive) subunit. Chondrogenic differentiation inducing medium was defined as Dulbecco's modified Eagle medium-high glucose (DMEM-HG) supplemented with 10 ng/ml recombinant human TGF-. beta.1 (Merck, Darmstadt, Germany), 100 nM dexamethasone (Sigma), 0.1 mM L-ascorbic acid 2-sulfate (Fluka, St. Louis, MO, USA), 6. mu.g/ml insulin (Merck), 6. mu.g/ml transferrin (Sigma), 1 mM sodium pyruvate (Gibco, Grand Island, NY, USA), 0.35 mM L-proline (Merck), and 1.25 mg/ml Bovine Serum Albumin (BSA) (Sigma). The medium was periodically changed every 3 days for 3 weeks. At days 7 and 21 after differentiation, approximately 10 microspheres were harvested and histologically evaluated, including conventional H & E staining, type II collagen immunohistochemistry and glycosaminoglycan (GAG) analysis, biochemical evaluation to determine GAG/Hydroxyproline (HYP) ratio, and mechanical evaluation of the elastic modulus of chondrogenic differentiated microspheres. These microspheres (which are cartilage-like functional subunits) are then used for the integration and assembly steps of complex tissue engineering.

Cartilage-like functional subunits stained positively for Alcian blue (Alcian blue), indicating the presence of glycosaminoglycans and type II collagen. Although the GAG/HYP ratio for native cartilage was only a value of 7%, the composition and structure of the micro-substances improved when they were further cultured in co-culture medium for 14 and 21 days, which increased by 12 and 15% of the native tissue, respectively, indicating that further engineering in co-culture medium was ongoing. Longer incubations and stimulation with further chondrogenic differentiation signals may be used.

Example 5: manufacture of bone-like functional subunits

Osteogenic differentiation of rMSC in collagen microspheres was induced by culturing rMSC-collagen microspheres as a suspension in osteogenic differentiation induction medium 3 days after formation of the naive (nadive) subunit. Osteogenic differentiation induction medium was defined as Dulbecco's modified Eagle's medium-Low glucose (DMEM-LG) supplemented with 10% FBS, 100 nM dexamethasone (Sigma), 0.1 mM L-ascorbic acid 2-phosphate (Fluka, St. Louis, MO, USA) and 10mM beta-glycerophosphate (Sigma). The medium was periodically changed every 3 days for 3 weeks. At 7 and 21 days post-differentiation, approximately 10 microspheres were harvested and histologically evaluated, including conventional H & E staining, von Kossa staining, biochemical analysis of total calcium content of osteogenic differentiated microspheres.

These bone-like functional subunits stained positive with Von Kossa. Quantitative calcium assay showed that calcium levels reached more than 20% of the functional subunits (fig. 3C). Calcium deposition continued to increase with increasing co-culture time, indicating that the co-culture conditions successfully maintained the osteogenic phenotype. Co-culture involves culturing undifferentiated MSCs in the middle layer after combining into three layers in the appropriate configuration, which may also be naive (naval) subunits, osteogenic subunits and chondrogenic subunits, all together.

Example 6: quantitative analysis of GAG/HYP ratio of cartilage-like microspheres

GAG content was determined by the 1, 9-dimethylmethylene blue dye binding assay (Barbosa, et al,Glycobiolog, 13(9):647-53 (2003)). Briefly, for each set, 80 microspheres of each sample were placed in a pH 6.5 phosphate buffer (50mM phosphate buffer, 5mM EDTA and 5mM L-cysteine) containing 300. mu.g/mL papain for solubilization of proteoglycans. Digestion was carried out overnight at 60 ℃. The absorbance at 656 nm was then determined using a microplate reader (Safire 2;. Tecan, Mannedorf, Switzerland). The amount of GAG in the sample was determined with a calibration curve prepared using a linear region of chondroitin sulfate between 0.5-2 μ g/100 μ L as a standard (Barbosa et al,Glycobiology, 13:647-653 (2003)。

collagen content was determined by the Sircol collagen test. Briefly, for each set, 80 microspheres of each sample were placed in a 0.5N acetic acid solution containing pepsin. Digestion was performed overnight at 4 ℃. The absorbance at 555nm was then determined with a microplate reader (Safire 2;. Tecan, Mannedorf, Switzerland). The amount of collagen in the sample was determined using a calibration curve prepared from a linear region between 6.25-25 μ g/100 μ L of bovine collagen standard. The hydroxyproline content was 14% of the total collagen content. The GAG/HYP ratio is shown, for example, in FIG. 3B. Example 7: different subunits were integrated for the fabrication and co-culture of double and triple layer osteochondral constructs.

Three hundred and sixty osteogenic differentiated collagen-rMSC microspheres were packaged. Just before packaging three hundred sixty soft bone forming differentiated collagen-rMSC microspheres as an upper layer, 2mg/ml with 5x10⁵An aliquot of 200 μ l collagen gel of undifferentiated rMSCs per ml of cells was added to the intermediate layer. The three layers of osteochondral constructs were then divided into 3 groups and cultured in Normal Medium (NM), Chondrogenic Medium (CM) or Osteogenic Medium (OM) or for another period of 14 or 21 days. In a similar manner, a two-layer construct consisting of chondrogenic or osteogenic parts together with an undifferentiated rMSC-collagen gel layer was made and used as a control. All co-cultured constructs were evaluated histologically, histochemically and immunohistochemically with GAG and type ii collagen as markers for cartilage formation, calcium deposition as markers for osteogenesis, and type X collagen as markers for linkers. The ultrastructure of the trilayer constructs was also evaluated by Scanning Electron Microscopy (SEM) and energy scattering X-ray (EDX) analysis. The micro-material comprising the entire construct is still visible.

Example 7: histological and immunohistochemical evaluation

Samples (whole construct after co-cultivation, or tri-or bi-layer construct) were fixed in 4% Paraformaldehyde (PFA), embedded in paraffin and cut into 7 μm pieces. Hematoxylin and eosin (H & E) staining was used to show cell morphology, safranin O staining was used to show GAG rich areas, immunohistochemistry for type II collagen and type X collagen were used as phenotypic markers for chondrocytes and hypertrophic chondrocytes, respectively.

For type II collagen immunohistochemistry, sections were incubated with 0.5% pepsin at 5mM HCl for 30 minutes at 37 ℃ for antigen retrieval. After incubation overnight at 4 ℃ with mouse anti-type II collagen polyclonal antibody (Calbiochem) diluted 1:2000 in PBS, sections were incubated with anti-mouse secondary antibody diluted 1:200 in PBS for 30 minutes at room temperature.

For immunohistochemistry against type X collagen, sections were incubated with 0.2% hyaluronidase in PBS for 1 hour at 37 ℃ and then with 0.1% pronase (in PBS) for 9 minutes for antigen retrieval. After overnight incubation at 4 ℃ with mouse anti-type X collagen monoclonal antibody (Abcam) diluted 1:2000 in normal horse serum, sections were incubated with anti-mouse secondary antibody diluted 1:800 in normal horse serum for 30 minutes at room temperature. The Vectastain ABC kit (Vector Laboratories) and the DAB substrate system (Dako) were used for color development in both cases according to the supplier's instructions.

Von Kossa staining was used to identify calcium deposition in mineral areas. Briefly, sections were immersed in 1% silver nitrate solution (Sigma) under UV irradiation for 30 minutes. Unreacted silver was removed by 2% sodium thiosulfate solution for 5 minutes. Nuclear Fast Red (Nuclear Fast Red) was used as the counterdye.

The appropriate zonal tissue with calcified cartilage junctions separating the osteoid and chondroid layers was identified in a three-layer configuration. Positive Von Kossa staining identified calcium deposition, positive type II collagen and positive alcian blue staining indicated cartilage, and positive type X collagen indicated hypertrophic nature of the three-layered structure. In contrast, no such compartmentalized tissue was identified in the bilayer configuration (control) or when the trilayer configuration was constructed without undifferentiated subunits in the middle layer. In the bilayer control, there is no intermediate layer; in the three-layer control (i.e., three layers without cells), pure collagen gel constituted the middle layer.

Example 8: quantification of calcium content

To assess whether the osteogenic phenotype was maintained in the chondrogenic differentiation medium during trilayer culture, calcium deposition from the osteogenic layer of microspheres was extracted with 1% trichloroacetic acid for 24 hours and quantified using a calcium assay kit (Bioassay Systems, Hayward, CA, Cat #: DICA-500). Briefly, the same volumes of reagent a and reagent B were combined and equilibrated to room temperature prior to use. Preparation of Ca by serial dilution in distilled water²⁺The standard solution of (4) (12.5-200. mu.g/mL). Aliquots of 5 μ L of the standard or sample are transferred to wells of a bottom clear 96-well plate and 200 μ L of working reagent is added. The mixture was incubated at room temperature for 3 minutes before measuring the absorbance at 612 nm. Determination of Ca present in samples by calibration against the linear region of the standard curve²⁺The amount of (c). As shown in fig. 3C, calcium deposition in the bone-like functional subunit is one tenth of that in native cartilage. As the time of co-culture increased, calcium deposition in the co-culture continued to increase, indicating that the co-culture conditions maintained and further supported the osteogenic phenotype.

Example 9: SEM and EDX analysis of osteochondral constructs and native osteochondral plugs (plugs)

To examine the microstructure of the three-layer simplices, the samples were processed for Scanning Electron Microscopy (SEM) analysis. The samples were rinsed with phosphate buffered saline and fixed in 2.5% glutaraldehyde for 2h at 4 ℃. After dehydration by a gradient series of ethanol, the trilayer constructs were dried at the critical point and fractured to expose their cross-sections. The samples were mounted on a holder with carbon resin and sputter coated with gold-palladium prior to inspection. The samples were then examined for microstructural analysis (data not shown) and for the detection of calcium, phosphorus and their relative distribution in the samples with an SEM (LEO 1530; LEO Electron Microscopy, Cambridge, UK) combined with energy scattering X-ray (EDX) spectrometry. The amounts of calcium and phosphorus were measured and the molar ratio of calcium to phosphorus was calculated (table 1). The calcium to phosphate content and their molar ratio suggest that the calcium content at the junction is high, while the content in the uncalcified cartilage formation or cartilage layer is negligible. This again validates the compartmentalized organization of the osteochondral graft. Furthermore, the fibrous morphology of the fibrous structure in the different layers is also consistent with other histological and biochemical evaluations, since many matrix materials including GAGs can be found deposited on the collagen fiber network in the cartilage layer, while many calcium phosphate particles or deposits can be found around the collagen fibers in the bone layer. Most importantly, many well-aligned collagen bundles or fibers could not be identified in the calcified cartilage joints, which has been characterized by vertically aligned collagen fibers.

Example 10: SEM image preprocessing and analysis of collagen fiber alignment by Radon transformation

MATLAB software was used to correct for non-uniform illumination (non-uniform illumination) and to increase the contrast of the SEM images before further analysis. In short, the grayscale SEM image is first converted to a binary image. The binary image is then skeletonized (skeletionization) prior to Radon conversion. In MATLAB, the image can be viewed as a matrix of intensities. The Radon transform is the projection of image intensity along a radial line in a direction of a particular angle theta. By using the Radon transform, θ is specified as 0-179 degrees, resulting in a value of 180 representing the magnitude of the straight line in the direction of that particular angle for each of the 180 angles. An intensity plot (intensity plot) is then generated for each angle so that the direction of the fiber can be found.

The variation of the peaks showing whether there is a preferred alignment is shown in table 2 below.

Table 2: mean difference in 95% confidence intervals for all or the first 50% peaks with intensity above the median in different layers of osteochondral grafts

The results show that the peak angle variation in the joint area is small, suggesting a preferred angle for the fiber or bundle. This further validates the scanning electron micrographs.

Example 11: BrdU labeling of rMSC

To track the undifferentiated MSCs during linker formation, the middle undifferentiated rMSC-collagen gel layer was labeled with 5-bromo-2-deoxyuridine (BrdU) before combining it with the pre-differentiated layer. The three layers of the construct were then cultured for 14 days before analysis. After the three layers of culture were completed, the constructs were fixed and BrdU immunohistochemistry was performed to follow whether the linker cells were differentiated from undifferentiated rmscs. Briefly, sections were incubated with 2N HCl at 37 ℃ for 30 minutes for antigen retrieval. They were then neutralized with sodium tetraborate solution for 10 minutes. After overnight incubation at 4 ℃ with mouse anti-BrdU monoclonal antibody (clone BMC 9318; Roche) diluted 1:1000 in PBS, sections were incubated with anti-mouse secondary antibody diluted 1:800 in PBS for 30 minutes at room temperature. The Vectastain ABC kit (Vector Laboratories) and the DAB substrate system (Dako) were used for color development in both cases according to the supplier's instructions. Whether BrdU labeling interacts with the differentiation process was also investigated by incubating BrdU labeled MSCs with normal media for 1, 7 and 21 days or with chondrogenic media for 21 days.

BrdU-labeled undifferentiated MSCs localized at the junction region, suggesting that the presence of undifferentiated MSCs in the middle layer contributed to at least the formation of hypertrophic chondrocytes at the junction.

Example 12: secretion of BMP2 from osteogenic differentiated progeny of MSCs in osteoid functional subunits

Using mouse MSCs and collagen, different sizes of osteogenic functional subunits were prepared as described in example 5. Constructs prepared from 2.5, 10, 50 and 150 μ L droplets were prepared and cultured in osteogenic differentiation medium for 21 days. During this 3 week period, conditioned media was collected from these functional subunits and analyzed for bone morphogenic protein 2(BMP2) by ELISA. The amount of BMP2 in the collected medium was determined against a calibration curve. The increase in secretion of BMP2 over time from different sized bone-like functional subunits is shown in figure 4. The results indicate that BMP2 secretion increases with increasing culture time, suggesting that osteogenically differentiated progeny of MSCs present in the osteogenic subunits continuously synthesize and secrete BMP 2. Since BMP2 is able to stimulate chondrocyte hypertrophy, factors, including BMP2, likely secreted from the osteogenic functional subunit (bottom layer) induce differentiation of the undifferentiated MSCs of the middle layer towards a calcified cartilage phenotype.

Example 13: osteochondral repair by layers of functional subunits

In these experiments, new zealand white rabbits with mature bones (>5 months old) were used. Bilateral knee joints from the same rabbits were randomly divided into experimental and control sides. After general anesthesia, medial paraspinal incisions were made on both lateral knee joints. The incision is continued until the medial femoral condyle is exposed. A full thickness osteochondral defect of 4 mm in diameter and 4 mm in depth was created in the medial femoral condyle using a sterile punch. During surgery, functional subunits of osteogenesis and chondrogenesis prepared as described above are placed aside. A plurality of osteogenic functional subunits are implanted to press-fit (press-fit) the defect, leaving a thin layer on top. A plurality of chondrogenic functional subunits are then placed on the osteogenic layer and the defect is pressed in again. And (5) pressing defects by using a spatula. The implanted construct is positioned in the defect by protecting it with a layer of collagen gel under the photochemically cross-linked collagen film and securing it by suturing. On the control side, the defect is empty. Wound healing takes place in the layers. After surgery, the rabbits were allowed to move freely. One month after implantation, the general appearance of the defect was examined and recorded. A full thickness osteochondral biopsy was taken for fixation and decalcification, followed by histological, histochemical, and immunohistochemical analysis.

Superior compartmentalization of the cartilage region and the layer with the wave tide marked calcified cartilage junctions separating the cartilage and subchondral bone layers was found. In contrast, defects in natural healing (the control discussed above) and repair with undifferentiated or naive (nave) subunits still show a large amount of fibrous tissue with no zoning tissue at this point in time.

Example 14: osteochondral repair by in vitro regenerated three-layered osteochondral construct with appropriate compartmentalized tissue

In these experiments, full thickness osteochondral defects were generated in new zealand white rabbits (>5 months old) with mature bones as described in example 14. The rMSCs were isolated and cultured as in examples 1 and 2. Three-layered osteochondral constructs with intact calcified joints and thus appropriate compartmentalized tissue were prepared using autologous cells and using the procedures described in examples 3-5 and 7 and injected to press fit defects. An aliquot of collagen gel was applied to the surface of the construct and a photochemically cross-linked collagen membrane was used to protect the implant with a single suture. The control side treatment, other procedures, sample harvest time and preparation methods were the same as described in example 14.

Superior compartmentalized organization of cartilage regions and layers with calcified cartilage junctions separating the wave tide marks of cartilage and subchondral bone structures was found. In contrast, natural healing (the control discussed above) still showed a large amount of fibrous tissue with no zoning tissue at all at this time point.

Example 15: co-culturing osteochondral grafts in compression-based bioreactors

The rMSCs were isolated and cultured as in examples 1 and 2. A volume of 70 μ L of naive (naive) subunits was prepared as described in example 3. Cartilage-like and bone-like functional subunits were produced with cylindrical and 100 μ L volumes, respectively, as described in examples 4 and 5, for 21 days. The functional subunit and naive (nameive) subunit are combined at the end of 21 days to form a complex tissue with heterogeneous tissue components. These structures were mechanically compressed using a bioreactor setup with 10% peak-to-peak replacement, 0.5Hz, 1 hour per day, for a 21 day protocol while being cultured in chondrogenic differentiation medium as described in example 4. The complex tissue-like structures were then processed for histological, histochemical, and immunohistochemical evaluations as described in example 8. Histological evaluation of the samples showed that the linker region was positive for GAG, type II collagen as markers for cartilage, and positive for von kossa staining as markers for calcification. For all constructs co-cultured in a compressed bioreactor, the linker is intact and the entire construct is stable and steerable. On the other hand, some constructs that were not exposed to the compression-based bioreactor co-culture failed to maintain integrity, with the different layers dispersed. After tensile strength testing of intact constructs surviving during co-cultivation, the linker strength was much higher in those with chondrogenic co-cultivation media than in normal media, while in those with compression during the loading period, the linker strength was also higher than those without compression especially at normal media and later time points (fig. 5).

Example 16: control of cell alignment using torsion-based bioreactors (cell alignment)

The rMSCs were isolated and cultured as in examples 1 and 2. 1000 μ l of bone-like subunits were made on gear-like polycarbonate rods as described in example 5 for 21 days. A 3000 μ l hollow tube or thin layer subunit was fabricated between two bone-like subunits as described in example 5 using a custom made container consisting of a smaller inner cylinder inserted in the center of a large outer cylinder. The lamellar subunits are formed by filling the cavity between the outer and inner cylinders with a cell-matrix mixture. After removal of the outer cylinder, the complex tissue consisting of two bone-like subunits and thin-layer subunits was mounted to a bioreactor and loaded with torque with a protocol of 15 degrees at 0.5Hz and 2 hours per day for 14 days while culturing in normal medium set up using the bioreactor. The control structure was exposed to static loading by simply clamping the structure onto the bioreactor. The structure was incubated in normal medium using a bioreactor for the same period of time. The complex tissue structure is then processed for histological evaluation as described in example 8. Image analysis software based on MATLAB was used to assess cell alignment in the constructs. The results show that the cell orientation is a preferred angle of about 45 ° in those under cyclic torsional loading, and about 100 ° (along the long axis of the bioreactor) in those under static loading (table 3). Multiple thin layers can be used to create cell alignment at different preferred angles, a structure that is important for tissues such as the annulus fibrosus of an intervertebral disc.

TABLE 3 alignment of cells under static and cyclic torsional loading

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The publications cited herein and the materials to which they are cited are specifically incorporated by reference.

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 herein. The following claims are intended to cover such equivalents.

Claims (21)

1. A method of preparing a bioengineered tissue graft comprising

Combining two or more functional subunits, optionally separated by undifferentiated encapsulated pluripotent or multipotent cells, wherein the two or more functional subunits each comprise encapsulated pluripotent or multipotent cells that can be induced to differentiate into different cell types; and

the combined functional units are cultured to form a bioengineered tissue graft that mimics the structural and functional characteristics of complex tissues.

2. The method of claim 1, wherein the pluripotent or multipotent cells are selected from the group consisting of induced pluripotent stem cells, embryonic stem cells, fetal stem cells, cord blood stem cells, bone marrow-derived stem cells, and adipose tissue-derived stem cells.

3. The method of claim 1, wherein the functional subunit comprises an extracellular matrix biomaterial.

4. The method of claim 1, wherein the extracellular matrix biomaterial comprises collagen.

5. The method of claim 1, wherein the functional subunit is selected from the group consisting of a microsphere, a cube, a ring, or a nanorod.

6. The method of claim 1, wherein the cells are induced to differentiate using a method selected from the group consisting of chemical induction, genetic manipulation, and remodeling of biological and mechanical microenvironments.

7. The method of claim 1, wherein the complex tissue is selected from the group consisting of osteochondral tissue, intervertebral discs, spinal motion segments; ligamentous bone tissue, a cardiac band having cardiac muscle containing a network of blood supplies; a highly organized layered growth plate with cartilage capable of lengthening and thickening bone tissue and a liver patch with fully blood-supplied hepatocytes.

8. The method of claim 1, wherein the combined functional units are cultured in the presence of conditioned media, growth factors, cytokines, serum, and combinations thereof.

9. The method of claim 6, wherein the chemical conditions are selected from the group consisting of antioxidants, acids, bases, oxygen tension, and combinations thereof.

10. The method of claim 1, wherein the combined functional units are cultured in the presence of a force selected from the group consisting of torsional force, compression, tension, and combinations thereof.

11. The method of claim 1, wherein the complex tissue is osteochondral tissue comprising compartmentalized tissue.

12. The method of claim 1, wherein the complex tissue is a spinal motion segment comprising a bone block, a cartilage endplate, a nucleus pulposus, and a fibrous ring structure.

13. The method of claim 1, further comprising fine-tuning the structural and functional properties of said bioengineered tissue graft by exposing said bioengineered tissue graft to suitable biological, chemical and physical co-culture conditions.

14. A method of repairing tissue damage in a subject comprising

Administering the bioengineered tissue graft produced by the method of claim 1 to a site of tissue damage in a subject.

15. A bioengineered tissue graft produced by the method of claim 1.

16. A method of producing a calcified regional junction in a bioengineered tissue construct comprising

Culturing in vitro a construct having first, second and third layers to induce a calcified region between the first and third layers;

wherein the first layer comprises microencapsulated pluripotent or multipotent cells that can be treated to induce osteogenic differentiation;

wherein said second layer is adjacent to said first layer, separates said first and third layers, and comprises undifferentiated microencapsulated pluripotent or multipotent cells;

wherein the third layer is adjacent to the second layer and comprises pluripotent or multipotent cells that can be treated to induce chondrogenic differentiation.

17. A bioengineered tissue construct comprising a first, second, and third layer;

wherein the first layer comprises microencapsulated pluripotent and multipotent cells that can be treated to induce osteogenic differentiation;

wherein the second layer is adjacent to the first layer, separates the first and third layers, and comprises undifferentiated microencapsulated pluripotent or multipotent cells;

wherein the third layer is adjacent to the second layer and comprises pluripotent or multipotent cells that can be treated to induce chondrogenic differentiation.

18. The bioengineered construct of claim 17, wherein the construct comprises a calcified regional linker between the first and third layer.

19. A method of treating osteochondral injury in a subject, comprising administering the bioengineered tissue construct of claim 17 to the subject at the site of osteochondral injury.

20. A method of producing a complex tissue construct comprising

Culturing two layers of encapsulated stem cells that can be induced to differentiate under conditions that induce the formation of a complex tissue construct, the two layers of encapsulated stem cells being linked together by a layer of undifferentiated encapsulated stem cells.

21. A method of producing a bioengineered spinal motion segment comprising

Inducing microencapsulated pluripotent or multipotent stem cells to form osteogenic subunits;

combining osteogenic subunits with microencapsulated pluripotent or multipotent stem cells that can be induced to form chondrogenic subunits, wherein the combination of osteogenic subunits and chondrogenic subunits form a bone block comprising an endplate; and

an intervertebral disc having an annulus fibrosus encapsulating a nucleus pulposus is sandwiched with bone pieces comprising endplates to form a spinal motion segment.