WO1996039811A1 - Immunocompromised animals comprising human synovial tissue - Google Patents

Abstract

Immunocompromised hosts are provided, comprising a functional human synovial sac and articular cartilage. An intact human fetal patella is introduced into the xenogeneic host at a site that permits survival of the tissue, and permits experimental manipulation.Engrafted patella show early differentiation into a columnar matrix and stratification consistent with joint cartilage. The patellar articular surface is closely overlaid with a single layer of large cuboidal to attenuated cells with a central, oval to brick shaped nuclei and clear to pale eosinophilic cytoplasm consistent with synoviocytes. This layer reflects on itself at the margins of the patellar graft and closes, forming a complete envelope over the patellar articular surface. The human articular tissue is useful for investigating the effects of agents such as bacteria and bacterial peptides, cytokines and proteases on synovial function and dysfunction, growing neoplastic synovial sarcoma tissue in a natural environment, studying the hematopoietic interactions that are involved in various inflammatory arthritic diseases, and determining the role of HLA haplotypes in development of joint diseases.

Description

Im unocompromi sed animal s compri sing human synovial ti ssue

The field of this invention is immunocompromised mammals comprising human synovial tissue.

Human skeletal movement is made possible by 7 classes of freely moving (synovial) joints, and by 6 classes of fixed (cartilaginous) joints. Within the 264 synovial joints is an even greater number of articulating surfaces. A synovial joint is a functional unit that allows 2 or more opposed limb bones to move freely in relation to each other. The joint between the bones is cushioned by cartilage, and by the synovial sac. Within the synovial sac is synovial fluid, a clear viscous liquid that provides lubrication and nourishment for the cartilage. Synovial fluid is produced by dialysis of plasma across the synovial membrane, which is then combined with a hyaluronate-protein complex secreted by the synovial membrane.

There are a number of diseases known in humans that affect the joints, and particularly the synovium. These include synovial sarcomas, osteoarthritis, bacterial and fungal infections, inflammatory, autoimmune and hemorrhagic diseases. Combined, they are a cause of great pain and suffering in the population, with little effective therapy apart from symptomatic treatment with analgesics and antiflammatory drugs. Rheumatoid arthritis affects 1% of populations worldwide. There is significant immunological activity within the synovium during the course of the disease. It is believed that this reactivity provides an intense stimulus to the synovial lining cells, which then undergo a transformation into an invasive pannus that brings about joint erosion through the release of destructive mediators. The release of cytokines, proteases and reactive oxygen intermediates have all been implicated in the disease pathology. The initiating factor is unknown, but might be an infection, trauma, bacterial infection, or autoreactivity. There is increased risk of disease for persons having the HLA-Dw4 allele. The term "seronegative spondylarthropathies" is used for an entity of rheumatic syndromes of peripheral joints and the spine, such as ankylosing spondylitis, reactive arthritis (ReA), Reiter's syndrome, arthritis in psoriasis and in inflammatory bowel disease, which are strongly associated with expression of the MHC class I molecule HLA- B27. Reactive arthritis is triggered by infection, usually urethritis or gastroenteritis. Two recent observations have excited interest: first, antigen and DNA from the triggering bacteria have been identified in the joint and, second, ReA synovial T cells have been found to respond specifically to the bacterium that caused the initiating infection. This raises the possibility that the disease pathology could be studied in an experimental model.

The field of medicine relies heavily on animal models. Most arthritis models are induced diseases, such as collagen or adjuvant arthritis. Spontaneous murine models for arthritis are rare, and have been highly variable from one mouse colony to another. Other animal models, such as dog and rat rheumatoid arthritis models, have been found to be inconsistent in pathology. The known animal models for osteoarthritis do not accurately simulate the human disease, because none feature secondary changes, such as synovial proliferation. There are many shortcomings with these models. Since the diseased cells and tissue are not human tissue, it is uncertain whether the animal cells can provide a reasonable prediction for the human correlate. Furthermore, a number of cytokines and other mediators and factors are human specific. The study of these agents and the development of therapies are seriously hampered by the lack of suitable animal models.

Having viable human tissue in an animal model provides numerous advantages. One can investigate the effect of agents on the tissue at various stages in the development of the disease. The interactions of cells, secreted agents and tissue can also be analyzed. A xenogeneic animal model further provides a means of testing the effect of factors and other agents on cells that are difficult to maintain in culture. Short-lived lymphocyte subsets, neural cells, complex tissues, neutrophils, etc. that cannot easily be grown in culture for extended periods of time may be examined.

It is therefore of substantial interest to develop and provide animal models comprising human joint cartilage and synovium that remains viable for extended periods of time. Such an animal model would permit investigation of the changes in the tissue, the etiology of disease and the effect of agents on pathogens.

A description of the SCID-hu mouse may be found in J.M. McCune et al. (1988) Science 241:1632-1639; R. Namikawa et al. (1990) J. Exp. Med. 172:1055-1063 and J.M. McCune et al. (1991) Ann. Rev. Immunol. 9:395-429. SCID-hu mice having long bone and bone prϊmordia implants are described in published International patent application PCT/US92/06309, and European patent application no. 88.312222.8.

Immunocompromised mouse strains are described in S. Nonoyama et al. (1993) J. Immunol 150:3817-3824; I. Gerling et al. (1994) Diabetes 43:433-440; Bosma, et al. (1983) Nature 301:52; and P. Mombaerts et al. (1992) CeH 68:869-877.

Problems in joint pathology are reviewed by Gardner (1994) J. Anat. 184:465-76.

An overview of the pathogenesis of the HLA-B27-associated spondylarthropathies, particularly reactive arthritis, may be found in Hermann et al. (1994) Ann. Med. 26:365- 9; Kingsley and Sieper (1993) Immunol. Today 14:387-91; and McGuigan et al. (1985) Semin Arthritis Rheum 15:81-105.

The role of various cytokines and proteases in rheumatoid arthritis is discussed in Feldmann et al. (1994) Circ. Shock 43:179-84; and Testa et al. (1994) Clin. Orthop. 308:79-84.

Immunocompromised hosts are provided, comprising a functional human synovial sac and articular cartilage. An intact human fetal patella is introduced into the xenogeneic host at a site that permits survival of the tissue and permits experimental manipulation. The synovial primordium on the articular surface of the patella develops into a complete synovial sac. The chimeric animal has broad applicability in the study of degenerative and metabolic human articular diseases. The human articular tissue may be used for investigating the effects of agents such as bacteria and bacterial peptides, cytokines and proteases on synovial function and dysfunction, growing neoplastic synovial sarcoma tissue in a natural environment, studying the hematopoietic interactions that are involved in various inflammatory arthritic diseases, and determining the role of HLA haplotypes in development of joint diseases.

Methods and compositions are provided for the production of human articular tissue in an immunocompromised heterologous mammalian host, particularly a mouse, for extended periods of time. The method comprises implanting human fetal patella in an appropriate site in an immunocompromised host. By histologic examination, it was found that the articular surface of human fetal patella at ages 16 to 24 gestational weeks was partially covered by a single cell thickness of a cell population consistent with synovial cells, representing synovial primordium. The synovial cells on the patella surface were found to proliferate and differentiate. The developing synovial layer is found to reflect on itself and closes, forming an intact synovial sac that contains synovial fluid. The chimeric animal provides an easily manipulated experimental model that is useful for studying human articular diseases, particularly those with an inflammatory, genetic or infectious component.

The host animal is engrafted with a human fetal patella, including the bony tissue, cartilage and the cells found on the articular surface. The tissue is vascularized, except in the cartilage, which is normally avascular. The implant is able to survive and differentiate in the host, where the primordial synovial cells develop into a complete synovial sac containing synovial fluid. The term patellar tissue shall be used to designate the bone and adjoining soft and connective tissue that is implanted, and the tissue that is derived therefrom after growth in the host animal. The patellar tissue may be used after at least about 2 weeks and the tissue will remain functional for about 4 months or more. The chimeric animal provides an environment for the introduction of a number of agents that are suspected of causing or contributing to joint disease, as well as the appropriate antagonists and blocking agents. Immunocompromised mammalian hosts suitable for implantation and having the desired immune incapacity exist or can be created. The significant factor is that the immunocompromised host is incapable naturally, or in conjunction with the introduced organs, of mounting an immune response against the xenogeneic tissue or cells. Therefore it is not sufficient that a host be immunocompromised, but that the host may not be able to mount an immune response after grafting, as evidenced by the inability to produce functional syngeneic host B-cells, particularly plasma cells, and/or T-cells, particularly CD4⁺ and/or CD8⁺ T-cells after implantation. Of particular interest are small mammals, e.g. rabbits, gerbils, hamsters, guinea pigs, etc., particularly murines, e.g. mouse and rat, which are immunocompromised due to a genetic defect which results in an inability to undergo germline DNA rearrangement at the loci encoding immunoglobulins and T-cell antigen receptors.

Presently available hosts include mice that have been genetically engineered by transgenic disruption to lack the recombinase function associated with RAG-1 and/or RAG-2 (e.g. commercially available TIM™ RAG-2 transgenic), to lack Class I and/or Class II MHC antigens (e.g. the commercially available CID and C2D transgenic strains), or to lack expression of the Bcl-2 proto-oncogene. Of particular interest are mice that have a homozygous mutation at the scid locus, causing a severe combined immunodeficiency which is manifested by a lack of functionally recombined immunoglobulin and T-cell receptor genes. The scid/scid mutation is available or may be bred into a number of different genetic backgrounds, e.g. CB.17, ICR (outbred), C3H, BALB/c, C57B1/6, AKR, BA, BIO, 129, etc. Other mice which are useful as recipients are NOD scid/scid; SGB scid/scid, bh/bh; CB.17 scid/hr; NIH-3 bg/nu/xid and META nu/nu. Transgenic mice, rats and pigs are available which lack functional T cells due to a homozygous disruption in the CD3e gene. Immunocompromised rats include HsdHan:RNU-/7.u; HsdHan:RNU-r«w/+; HsdHan:NZNU-rn«; HsdHan:NZNU-rm./+; LEW/HanHsd-777_.; LEW/HanHsd-rnw/+; WAG/HanHsd-rnw and WAG/HanHsd-r««/+.

Additional loss of immune function in the host animal may be achieved by decreasing the number of endogenous macrophages before, during, or after implantation of the xenogeneic tissue. Of particular interest is the reduction of macrophages by administration of dichloromethylene diphosphonate (Cl-MDP) encapsulated in liposomes, as described in co-pending application no. 08/169,293.

The host will usually be of an age less than about 25% of the normal lifetime of an immunocompetent host, usually about 1 to 20% of the normal lifetime. Generally, the host will be at least about four weeks old and large enough to manipulate for introduction of the donor tissue at the desired site. For example, mice are usually used at about 4 to 6 weeks of age. Growth of the tissue within the host will vary with the organ.

The mammalian host will be grown in conventional ways. Depending on the degree of immunocompromised status of the mammalian host, it may be protected to varying degrees from infection. An aseptic environment is indicated. Prophylactic antibiosis for protection from Pneumocystis infection may be achieved for scid/scid mice with 25-75 mg trimethoprim and 100-300 mg sulfamethoxazole in 5 ml of suspension, given three days each week, or in impregnated food pellets. Alternatively, it may be satisfactory to isolate the potential hosts from other animals in gnotobiotic environments after cesarean derivation. The feeding and maintenance of the chimeric host will for the most part follow gnotobiotic techniques.

A suitable site for implantation must be able to accomodate the size of the implanted tissue, to provide for vascularization, and to permit experimental manipulation. Subcutaneous implantation is conveniently used. The tissue will be implanted, conveniently by incision of the host skin and placement with a trocar, etc.

Other tissues may be transplanted into the host in addition to the patella. For example, hematopoietic components may be included, such as stem cells, lymph nodes, embryonic yolk sac, fetal liver, pancreatic tissue, appendix tissue, tonsil tissue and the like, which may serve to provide human lymphoid and granulocytic cells in the immunocompromised host. Sites for introduction of additional tissue may include under the spleen capsule, abdominal wall muscle, under the renal capsule, in the anterior chamber of the eye, the peritoneum, the peritoneal lining, brain, subcutaneous, vascular system, spinal cord, membranous sacs or capsules of various tissue, the retroperitoneal space, reproductive organs, ear pinnae, etc.

Introduction of the secondary tissue may be achieved by injection, implantation, or joining blood vessels (and other vessels if necessary) of the donor and host, using intravenous catheters, trocars, and/or surgical incision, or the like. The tissue or cells of interest will generally be normal, e.g. non-transformed and non-malignant tissue or cells. With various organs one may include native surrounding tissue with the organ tissue itself. The surrounding tissue may comprise connective tissue, or portions of blood and lymphatic vessels. In some cases, whole organ grafts may be transplanted by anastomosing donor and host blood vessels, lymphatic vessels, and the like. For the most part, normal cells, tissue, and/or organs may be stably maintained and functional for at least about 3-6 months and frequently for at least about 10 months.

A mixed population of cells in suspension may be enriched for the particular cells of interest. For example, with bone marrow cells, the suspension may be enriched for T cells by Ficoll-hypaque density gradient centrifugation, fluorescence activated cell sorting, panning, magnetic bead separation, elutriation within a centrifugal field, or rosetting. In some instances it may be desirable to enrich cells by killing or removing other cells. This may be achieved by employing monoclonal antibodies specific for the undesired cells in the presence of complement or linked to a cytotoxic agent, such as a toxin, e.g. ricin, abrin, diphtheria toxin, or a radiolabel, e.g. ^1JII, or the like. Immunoaffinity columns may be employed which allow for specific separation of either the desired or undesired cells, depending on the antibody or fragment thereof used for selection and the nature of the mixture.

As appropriate, dispersed cells are employed, where the relevant organs are teased apart to yield viable cells in suspension. Cells of particular interest as a secondary implant are human hematopoietic cells, particular T cells, neutrophils, and other granulocytic and myeloid cells. Such cells may be obtained from an immunocompetent human donor. The hematopoietic cells may be mismatched as to HLA type with the patellar tissue, so as to provide a marker for the source, or may be matched as to HLA type in order to provide T cells that recognize antigen presented by the patellar tissue. The cells may be injected at the site of the patellar implant, optionally in conjunction with various cytokines, and the resulting effect on the synovial and cartilaginous tissue determined.

The human fetal patella may be fresh tissue, obtained within about 48 hours of death, or freshly frozen tissue, tissue frozen within about 12 hours of death and maintained at below about -10°C, usually at about liquid nitrogen temperature (- 120°C) indefinitely. Normally the tissue will not have been subject to culture in vitro for an extended period of time. The patella is provided as a whole bone, and will include such cartilage and synovial or primordial synovial cells as are normally present.

For the most part the donor tissue will be from a human fetus. Preferably, the tissue will be fetal tissue of from about 16 to 24 gestational weeks. Several human MHC alleles have been implicated in articular disease, and donor tissue having those haplotypes are of particular interest. HLA-B27 implants are of interest for the study of seronegative spondylarthropathies, such as ankylosing spondylitis and reactive arthritis. Implants having HLA-Dw4 (HLA-DRbl *04), particularly on both alleles, are of interest for the study of rheumatoid arthritis. A subset of osteoarthritis has been linked to a genetic defect in Type II collagen, where there is a substitution of a cysteine for arginine at position 519 of the protein.

The human articular tissue may be used for investigating the effects of agents such as bacteria and bacterial peptides, cytokines, proteases, anti-inflammatory drugs, etc. on synovial function and dysfunction, growing neoplastic synovial sarcoma tissue in a natural environment, studying the hematopoietic interactions that are involved in various inflammatory arthritic diseases, and determining the role of HLA haplotypes and other genetic factors in development of joint diseases. There are several classes of diseases that affect synovial joints. Some features of the disease pathologies are shared, while others are unique to a particular disease or syndrome. The broad categories are inflammatory, non-inflammatory, septic, hemorrhagic and malignant. Degenerative diseases are of particular interest. They may be inflammatory, as with seronegative spondylarthropathies, e.g. ankylosing spondylitis and reactive arthritis; rheumatoid arthritis; gout; and systemic lupus erythematosus; or non-inflammatory, as with the various manifestations of osteoarthritis.

The degenerative joint diseases have the common feature in that the cartilage of the joint is eroded, eventually exposing the bone surface. Destruction of cartilage begins with the degradation of proteoglycan, mediated by enzymes such as stromelysin and collagenase, resulting in the loss of the ability to resist compressive stress. Alterations in the expression of adhesion molecules, such as CD44 (Swissprot P22511), ICAM-1 (Swissprot P05362), and extracellular matrix protein, such as fibronectin and tenascin, follow. Eventually fibrous collagens are attacked by metalloproteases, and when the collagenous microskeleton is lost, repair by regeneration is impossible. Normally there is a balance of metalloproteases, which degrade collagen, and tissue inhibitors of metalloproteases (TIMPs), at least partially regulated by cytokines such as IL-lb (Swissprot P01584) and TNFa (Swissprot P01375). When there is a loss of this homeostatic control there is degradation of cartilage structure, starting a vicious circle in which inflammation and proteolysis enhance each other.

The pathology of degenerative joint diseases provides a number of points for examining therapeutic intervention. One may experimentally administer enzymes such as stromelysin and collagenase, the breakdown products of these enzymes, e.g. partially digested type II collagen, bone mineral dust, etc., or their antagonists, to determine whether degeneration is initiated. Alternatively, biomechanical stress may be put on the joint, and the resulting expression of these proteins determined. The expression of adhesion molecules may be followed immunohistochemically, or by in situ hybridization. Similarly, the expression of cytokines such as IL-lb and TNFa may studied, or exogenous cytokines may be administered. Human and mouse TNF and IL-1 are generally cross-reactive. The effect of these events on cartilage and synovial structure may be studied histologically. Inhibitors of metalloproteases, such as aprotinin, Frey's inhibitor, tranexamic acid and gabexate mesilate, have been experimentally administered to arthritis patients. The subject animal model provides a means to determine the therapeutic index of such drugs.

The inflammatory degenerative joint diseases have a hematopoietic involvement, where specific T cells and antibody complexes may be present, as well as neutrophils and other myeloid elements. The subject animal model provides a unique opportunity to manipulate the interactions between these immune cells, and the joint environment. Generally, the immune cells will be of human origin. T cells, particularly those from rheumatoid arthritis (RA), ankylosing spondylitis (AS) and systemic lupus erythematosus (SLE) patients may be administered to the host animal, and the effect on the synovium and cartilage examined. Neutrophils, particularly in combination with IL-8 (Swissprot PI 0145) or epithelial neutrophil activating peptide-78, which act as neutrophil chemoattractants, may be administered. Antibody producing cells, or immune complexes themselves, particularly rheumatoid factors, may be administered to the host.

Particular cytokines have been implicated in inflammatory joint disease, and may be administered alone or in combination with immune cells. TNFa, IL-la (Swissprot P01583) and IL-lb, IL-6 (Swissprot P05231), IL-8 and GM-CSF (Swissprot P04141) are all of interest. Steroidal and non-steroidal anti-inflammatory drugs, immunosuppressive drugs, such as FK506 and cyclosporin, and cytokine antagonists and blocking agents may also be administered in conjunction with an inflammatory disease model.

Evidence suggests that reactive arthritis (ReA) and rheumatoid arthritis may be initiated by an exogenous antigen. Epstein-Barr virus has been suggested as a causative agent in RA, as have a variety of Mycoplasma sp. Frequent triggering infections in ReA are Chlamydia trachomatis, Yersinia, Salmonella, Shigella or Campylobacter. The pathogenic organisms, antigenic fragments thereof, or immune cells from patients may be administered to the subject animals to determine the effect on the joint implant. For studying tumors, neoplastic cells, e.g. synovial sarcomas, are injected into the implant and allowed to grow. By having the neoplastic tissue present in the implant, one can determine the effect of the neoplastic cells on the proximal articular structure and by providing for other agents or organs, such as T cells, determine the ability of the normal cells to respond to the presence of the neoplastic cells. In addition, one may investigate how the neoplastic cells displace or destroy the normal cells. One may also study a wide variety of agents as to their effect on the tumor, as well as on the normal cells. Thus, one may develop a therapeutic index of the effect on normal cells as compared to the effect on the tumor. In this manner, drugs may be better evaluated as to their use in treating tumors while not having a seriously adverse effect on the surrounding tissue.

Various drugs may be administered to the host and analytical methods used to determine the effects of agents on human articular tissue by invasive or non-invasive techniques. Non-invasive techniques include NMR, CAT scans, fluoroscopy, roentgenography, radionuclide scanning, ultrasonography, electrocardiography, electroencephalography, evoked potentials, etc. Invasive techniques include biopsy, autopsy, laparotomy, intermittent intravenous blood sampling, or intravenous catheterization, etc. Convenient placement of various devices, e.g. catheters, electrodes, etc. may be performed for continuous monitoring. Standard analyses for synovial fluid include measurement of volume, color, viscosity, mucin clot, leukocyte count, neutrophils, glucose, and microbiologic culture. Histological sectioning and staining may be used for gross or microscopic inspection of the tissue. Histological staining may include Alcian blue for evaluation of cartilage ground substances, periodic acid Schiff for polysaccharides, and Masson's trichrome for collagen deposition, as well as other stains knwn in the art. Immunohistochemical techniques may be used to determine the presence of antigens of interest, including the expression of adhesion molecules and cytokine receptors. In situ hybridization may be used to detect specific RNA and DNA sequences. Thus, the host may be used to determine the cytotoxicity and efficacy of various agents on human articular tissue, the effect on growth and viability of various human tissues, the effect of combinations of compounds, e.g. drugs, or the like. The manner of administration will vary greatly, depending upon the nature of the drug. It may be provided orally, ad libitum, intraperitoneally, intravascularly, subcutaneously, intrathymically, or the like. Depending upon the nature of the agent, one may use continuous infusion, employing various pumps that are available, which allow for long-term constant or varying infusion of a reagent. Usually, different dosage levels will be employed, based on past experience with the drug, anticipated levels with human treatment, toxicity or side effects, experience with the particular chimeric host, and the like. The effect of the drug may be monitored for any convenient time, usually at least 1 week from the initiation of administration of the drug, more usually at least 2 weeks, and at times for periods as long as 6 weeks or more. Preferably, determinations will be made in the period from about 2-6 weeks. Each agent may be administered at varying concentrations to determine effective dosages, response to variation in dosage and the like. Dosages may vary from picograms to grams per kilogram of host. Other methods of administering the agent may be employed as appropriate.

Phenotyping of the human cells to verify their origin and stage of developmental progression may be performed by standard histological methods, by immunohistochemistry, antibody staining or in situ hybridization with RNA and/or DNA probes. The exact method is not critical to the invention, and will depend on the exact cell types being studied. HLA markers may be used to distinguish the established xenogeneic organ transplants. The HLA type can be readily determined by staining with an appropriate antibody directed against any of the alleles of the human HLA locus, including Class I antigens.

The following examples are offered by way of illustration and not by way of limitation.

EXAMPLE 1 :

The following procedures were undertaken to construct a biologically relevant chimeric mouse with intact, normal human synovial sac and articular cartilage. The presence of normal human articular and synovial tissues in the mouse can then be applied to disease induction to serve as an accurate model of human articular disease, with broad applicability in the discovery and development of novel pharmacologic and biotechnologic compounds.

By histological examination, it was found that fetal human patella at the gestational age of 16 to 24 weeks is partially covered on the articular surface by a single cell thickness of a cell population consistent with synovial cells, representing the synovial primordium. By implantation of the intact patella, together with this synovial primordium, in a subcutaneous location, all or part of the synovial primordium is encouraged to develop into a complete synovial sac overlying the presumptive articular surface and containing synovial fluid.

Methods

The host animals were 4 to 6 week old C.B-17 scid/scid immunodeficient mice of either sex. Intact legs from 16 to 24 gestational week human fetuses are received in appropriate shipping media. Using suitable precautions, the leg is removed from shipping media and placed in a dish of media. The patella is dissected broadly from the anterior aspect of the femurotibial joint, preserving adjoining soft and connective tissue with care not to traumatize the articular surface. The patella is maintained in media during transport and holding prior to implantation.

The mice are anesthetized with ketamine/xylazine cocktail by intramuscular injection, or methoxyfluorane (open-drop inhalation). The left lateral abdomen is clipped and cleaned for surgery. A short incision is made in the left flank through the skin and dermis perpendicular to the axis of the vertebral column. With gentle handling, the patella is removed from holding media and inserted through the incision into the subcutaneous space in a rostrad direction with the patella articular surface adjacent to the dermis. Once the patella has been located away from the incsion, the skin is closed with one or more stainless steel wound clips.

Animals are held in recovery and observed to rouse from anesthesia, and then checked every 24 hours for 3 days post-surgery. Starting at two weeks post surgery, animals may be examined for graft success. Pre-mortem screening

Starting at two weeks post-surgery, the patella can be gently palpated across the skin. At this time, the skin should slide over the graft surface with an oily, smooth response to digital pressure, indicating the presence of synovial fluid.

Terminal examination

After euthanasia, the left lateral abdominal wall is broadly dissected across both skin and abdominal wall to preserve the contents of the implant and to not disturb the synovial and articular surface. The entire tissue mass, including the patellar graft, is immersed in 10% phosphate buffered formalin for a minimum of 48 hours prior to sectionng. Once fixed, the patella is trimmed in a sagittal direction, leaving adjacent soft tissue intact. If the graft is of advanced age (more than 4 months post-surgery) decalcification may be required prior to trimming. To decalcify, the post-fixation tissue is soaked for 8-18 hours, depending on the size, in a solution of a dilute acid, such as 0.5% HCl and EDTA. A 3 to 4 mm thin sagittal section of the graft is then placed into a disposable cassette, immersed in 10% phosphate bufered formalin, and submitted for routine paraffin embedding, sectioning and staining.

Histopathologic Examination

Sectioned human patella with synovium in the context of surrounding mouse soft tissue can be examined with routine hematoxylin/eosin staining, or with other staining methods to better evaluate cartilage ground substances (Alcian Blue), polysaccharides (periodic acid Schiff) and/or collagen deposition (Masson's trichrome). Unstained sections, placed on poly-L-lysine coated slides can be used for immunostaining techniques.

Results

The grafts in the mice have been digitally palpated and found to be consistent with the presence of synovial fluid between the skin and the patellar articular surface.

Mice engrafted with normal fetal patella have been examined post-mortem at various time points. During dissection and gross sectioning of the tissue, a clear viscous fluid was present over the face of the patellar articular surface. Microscopically, the patellar bone is not ossified at less than 8 weeks post surgery, and is wholly composed of a stellate cartilaginous matrix. The destined articular surface is morphologically simple, with early differentiation into a columnar matrix and stratification consistent with joint cartilage. The patellar articular surface was closely overlaid with a single layer of large cuboidal to attenuated cells with a central, oval to brick shaped nuclei and clear to pale eosinophilic cytoplasm consistent with synoviocytes. This layer reflected on itself at the margins of the patellar graft, forming a complete envelope over the patellar articular surface. The microscopic emptiness of this closed envelope is consistent with the presence of synovial fluid, which is washed away during histologic processing.

The subject chimeric system transplants synovium in the context of adjacent human connective tissue and other supportive structures. The reflection and closure of a complete synovial sac provides a model for the treatment of degenerative arthritides, such as osteoarthritis, and other diseases and condition that affect the synovium and cartilage. After engraftment, the implanted human tissue can be manipulated in a systematic way. The consequences of such manipulations can be read out by various methods, as described. Modulation or manipulation of inflammatory mediators and cytokines, and their interactions with synovial cells, inflammatory cells, and the matrix compartment can conveniently be performed with the subject chimeric animals. The study of these interactions, and potential interventions, cannot currently be performed because of lack of an appropriate animal model.

Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be readily apparent to those of ordinary skill in the art in light of the teachings of this invention that certain changes and modifications may be made thereto without departing from the spirit or scope of the appended claims.

Claims

WHAT IS CLAIMED IS:

1. A non-human mammalian host lacking functional syngeneic B-cells and T- cells, comprising: a human fetal patella grown for at least two weeks in said host, wherein the synovial primordium on the articular surface of said human fetal patella develops into a complete synovial sac.

2. A host according to Claim 1, wherein said host is a mouse.

3. A host according to Claim 2, wherein said mouse has a homozygous mutation at the scid locus.

4. A host according to Claim 2, wherein said mouse lacks expression at least one of functional RAG-1 or RAG-2.

5. A method for producing a chimeric non-human mammalian host comprising a human patella with a complete synovial sac, said method comprising: implanting a viable normal human fetal patella in an immunocompromised non-human mammalian host lacking functional syngeneic B- and T-cells; and maintaining said host for a period of at least 2 weeks, wherein the synovial primordium on the articular surface of said human fetal patella develops into said complete synovial sac.

6. A method according to Claim 5, wherein said non-human mammalian host is a mouse.

7. A method according to Claim 6, wherein said mouse has a homozygous mutation at the scid locus.

8. A method according to Claim 6, wherein said mouse lacks expression of at least one of functional RAG-1 or RAG-2.

9. A method for determining the effect of an agent on human articular tissue, the method comprising: administering said agent to a non-human mammalian host lacking functional syngeneic B-cells and T-cells, comprising a human fetal patella grown for at least two weeks in said host, wherein the synovial primordium on the articular surface of said human fetal patella develops into a complete synovial sac to provide human articular tissue; and determining the effect of said agent on said human articular tissue.

10. A method according to Claim 9, wherein said non-human mammalian host is a mouse.

11. A method according to Claim 10, wherein said mouse has a homozygous mutation at the scid locus.

12. A method according to Claim 10, wherein said mouse lacks expression of at least one of functional RAG-1 or RAG-2.

13. A method according to Claim 9 wherein a pathogenic condition is induced in the human articular tissue, and the effect of the agent on the pathogenic condition is determined.