WO2006116530A2

WO2006116530A2 - Compositions and methods for treating pulp inflammations caused by infection or trauma

Info

Publication number: WO2006116530A2
Application number: PCT/US2006/015860
Authority: WO
Inventors: Helen Lu; Gunnar Hasselgren; Mona Mcalarney; Zhenni Zhou
Original assignee: The Trustees Of Columbia University In The City Of New York
Priority date: 2005-04-28
Filing date: 2006-04-28
Publication date: 2006-11-02
Also published as: WO2006116530A3

Abstract

Compositions are disclosed that include a matrix and pulp cell or cell that can become a pulp cell. Also included are methods for identifying such compositions, methods for using such compositions, e.g., for restoration of a damaged tooth, and kits that include such compositions.

Description

COMPOSITIONS AND METHODS FOR TREATING PULP INFLAMMATIONS CAUSED BY INFECTION OR TRAUMA

TECHNICAL FBELD This application relates to the field of tissue engineering, and more particularly to oral infection.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to provisional U.S. Application Serial No. 60/675,767, filed on April 28, 2005, which is herein incorporated by reference in its entirety.

BACKGROUND

Inflammations of tooth pulp are commonly treated by procedures that remove the tooth pulp or extraction of the entire tooth. This process results in devitalization of the tissue and permanent loss of the tooth. It is therefore desirable, especially in young individuals, to develop an approach that promotes preservation of the tooth and the vitality of its pulp. Clinical considerations for such an approach are 1) the inability of present dental materials to provide permanent microorganism-proof seals, 2) a paradigm shift from the current clinical treatment modality of removing the affected pulp in young teeth to pulp regeneration, which enables continued dentinal self repair, extended growth of immature teeth, continued ability to fight infection, and the reduction of complications due to prosthetic repair of endodontically treated teeth, and 3) the lack of viable pulp regeneration grafts. Despite the ability of dental pulp to self- regenerate and repair under specific conditions in vivo, the current endodontic treatment modality for affected young pulp is total or partial removal of pulp tissue. Pulp regeneration is generally not an aim of treatment. Rather, the goal is to prevent infection by sealing off the pulp space, and, in pediatric patients, too enable continued root growth. To summarize, the present treatment forms for inflamed dental pulps either remove the pulp or, in young teeth, are aimed at healing at the location of the pulp wound or closer to the root. There is no treatment form that is aimed at regaining pulp space lost to inflammation.

Current Treatments

Although widely practiced, pulpectomy (removal of pulp tissue) is not the only treatment option. The choice of clinical treatment depends on the nature of the pulp injury, age of the patient, and the presence or possibility of infection. If pulp damage is considered reversible, a form of vital pulp therapy may be utilized, where only minimal pulp tissue is removed

(pulpotomy or partial pulpotomy) or a wound dressing is placed directly on the exposed pulp (pulp capping). The coronal portion of the pulp space is then sealed and mineralization stimulated. The use of vital pulp therapies is particularly attractive for pediatric patients where the lack of continued tooth growth in pulpectomized teeth is problematic. Tooth fractures in immature teeth treated with pulpectomies occur at the bone level, thereby complicating or preventing restoration. Partial pulpotomy treatment, which is carried out at a level 1-2 mm under a pulp exposure, preserves the cervical area of the pulp thus making continued dentin production possible. This increases the hard tissue bulk also in the cervical area preventing fractures. In carefully controlled studies, the success rates are high (91-93%) for partial pulpotomy. Emphasis is on physically sealing the pulp space through a mineralized barrier formed by the pulp and the use of dental materials to prevent further damage. A limitation of present vital pulp therapies is that mineralization occurs only apically (towards the tip of the root) to the level of the vital pulp exposure. Regeneration of dentin or dentin like tissue coronal to the pulp wound does not occur and the dental anatomy must be restored with dental materials. No current dental material can adequately duplicate the natural tooth with respect to mechanical properties and micro-leakage seal. The required restorations also tend to be large which accentuates the limitations of dental materials.

Description of the Pulpodentin Complex and the Clinical Significance of Pulp Injuries

Approximately 24 million root canal therapies per year are performed in the United States (ADA Survey of Dental Practice, 5S99, 1999). With a very conservative average cost of $400 per treatment this represents $9.6 billion per year for endodontic treatment alone. The tooth structure then must be restored with available dental materials. The restorative costs are approximately 14.4 billion dollars for an average restoration cost of $600. Therefore the total cost is approximately 24 billion dollars per year. Moreover, this cost is greatly underestimated since it does not include retreatment costs for failed therapies. It is clear that pulp-related endodontic

' diseases pose a significant clinical and social challenge.

The human tooth is consisted of the pulp and three types of hard tissues (enamel, dentin, and cementum). The enamel is the outer hard layer in the coronal portion of the tooth that provides a cutting and grinding surface for chemical digestion of food. Cementum covers the root surface and is the interface between the tooth and the fibers that connect the tooth to alveolar bone. The pulp is a loose connective tissue surrounded entirely by the dentin except at the tooth root apex, and it is the vital part of the tooth, containing cells, blood vessels, nerves, fibrous matrix, and ground substance. Both dentin and pulp are anatomically and functionally integrated in the pulpodentin complex. The pulp-dentin border is lined by odontoblasts, which are derived from pulp and are responsible for the production of dentin and mineralized tissue. The primary role of the pulp is to support the odontoblast layer. The nature of this support can be subdivided into four categories: formative, defensive, nutritive, and sensory. The major role of the odontoblast is dentin formation and repair/regeneration. When a tooth is injured, odontoblasts form more dentin and thereby maintain the vitality of the tooth. Although the pulp space decreases in volume after dentin regeneration/repair, this vital tooth remains capable of self-repair through dentin mineralization, growth (in immature teeth), fighting infection through host immune response, and sensation. If dentin repair/regeneration is not successful the traumatized pulp becomes necrotic due to invading bacteria. The greatest clinical implication of non-vital pulp is a high infection rate resulting in periradicular disease, bone loss and the cessation of growth for immature teeth. Depending on several factors, failure rates of 3-60% are reported for endodontic procedures. Most endodontic procedures are initiated because of pulp inflammation caused by infection stemming from caries. The treatment in the majority of cases involves pulpectomy. Pulpectomy is the removal of all pulp tissue, where the pulp space is cleaned, shaped, disinfected, and filled with a synthetic material. The tooth is then restored, often with a core build-up and a crown to replace tooth structures and function. The tooth is non-vital yet the structure of the tooth remains and the negative effects of tooth extraction are avoided. Nevertheless the treatment is both complex and time consuming. The technical complexity is reflected in the difference in failure rates between general dentists (30-35%) and endodontic specialists (5-15%) (Friedman, hi: Orstavik and Pitt Ford, (eds.): Essential Endodontology: Prevention and Treatment of Apical Periodontitis. Oxford, Blackwell Science, 1998). If a low 20% failure rate is assumed, the re- treatment cost is at least 4.8 billion dollars. The total re-treatment cost is higher since the failure rate of retreated teeth is significantly higher than the first treatment. Also increased damage to the tooth and supporting structures often occurs, thereby complicating treatment and increasing costs. Non-quantifiable costs such as time spent and pain are not included. Clearly an important limitation of current treatment is the high failure rate. One of the causes of failure is the complexity and sensitivity of the treatment technique. Another cause is the nature of the treatment. When all pulp tissue is removed the tooth is susceptible to infection for several reasons. One reason is that current dental materials cannot adequately seal the apical and/or coronal ends of the pulp space. Dentin cannot regenerate/repair without pulp support. Bacteria entering the pulp space have a rich nutritive source as they demineralize dentin. And these bacteria are almost impossible to treat since the pulp immune response no longer exists and systemic antibiotics cannot reach the pulp space.

Vital pulp therapies (pulp capping, partial pulpotomy, and pulpotomy) leave live pulp tissue in the tooth. Still, the pulp capping procedure has an increased failure rate with increasing time. The other two methods have a better outcome, but the pulp space lost to infection/inflammation is not regained with these procedures. A further limitation of vital pulp therapies is that mineralization occurs only apically to the level of the vital pulp. Regeneration of dentin or dentin like tissue coronal to the pulp wound does not occur and the dental anatomy must be restored with dental materials. No current dental material can adequately duplicate the natural tooth with respect to mechanical properties and microleakage seal.

SUMMARY

The present invention relates to methods and compositions for restoring a diseased or damaged tooth such that infection is inhibited or eliminated and pulp regeneration is facilitated. In general, the invention encompasses compositions and methods that include 1) a matrix (e.g., hydrogel matrix) with pulp cells or stem cells that support new tissue formation in the matrix and pulp cell infiltration, 2) the matrix further containing at least one antibiotic incorporated into the matrix, 3) the matrix containing antibiotic, the antibiotic being incorporated into a delivery vehicle such as a degradable polymer-based microsphere, the vehicle being embedded in the matrix, 4) the matrix further containing at least one angiogenic factor that is incorporated into the matrix, 5) the matrix containing at least one angiogenic factor, the angiogenic factor being incorporated into a delivery vehicle such as a degradable polymer-based microsphere, and the vehicle being embedded in the matrix, 6) the matrix containing at least one angiogenic factor, the angiogenic factor being incorporated into an aligned degradable polymer-based nanofiber mesh (e.g., PLGA), the mesh being embedded in the matrix. Accordingly, the invention also includes a composition comprising a physiologically acceptable matrix seeded with pulp cells. The matrix can include at least one agent that is an antibiotic (e.g., ciprofloxacin, Minicyclin, and metronidazole), antifungal agent, or growth factor; at least two antibiotics; or any combination thereof. In some aspects of the invention, the composition the agent is time released (i.e., an extended release composition). The matrix can be capable of being injected into the pulp chamber of a tooth. In some embodiments, the matrix of a composition includes a hydrogel (e.g., collagen, chitosan, alginate, MATRIGEL™, gelatin, JELL- O^®, fibrin), a mesh (e.g., polylactide-coglycolide (PLGA) mesh, polylactide (PLA) mesh, or polyglycolide (PGA) mesh, a cross-linked fiber mesh, a nanofiber mesh, a mesh fabric, biodegradable polymer mesh), a microsphere (biodegradable polymer microsphere, a hydrogel microsphere), or a combination of any of the foregoing, hi yet other embodiments, the matrix includes a nanofiber, an artificial three-dimensional scaffold material, or a synthetic three-dimensional scaffold material. The matrix can include a polycaprolactone polymer, a polygalactan polymer, a polyanhydri.de polymer, or a combination of any of the foregoing. Certain aspects of the invention include a matrix that includes type I collagen and type III collagen, e.g., in a ratio of type I collagen to type

HI collagen is 30%:70%, 55%:45%, 45%:55%, or 70%:30%. In other aspects of the invention, the matrix includes type I collagen (e.g., the collagen concentration is about 0.3% to 3.0%, about 0.3% to 0.5%, or about 0.5% to about 3.0%). The gelation pH of the collagen matrix can be about 6.0, 7.5, or 9.0.

In another embodiment of the invention, the composition includes alginate, and the alginate concentration is e.g., about 1.0% to 5.0%, 1.0% to 3.0%, or 3.0% to 5.0%. A composition can include CaCl₂ (e.g., at a concentration of about 50 mM, about 100 mM, or about 200 mM).

In some embodiments, the matrix includes chitosan.

In certain compositions, the viscosity of the gel is less that 100,000 cP at 37°C. The matrix of certain compositions can, in some cases, forms a scaffold upon which the pulp cells can grow.

A composition can include one or more cell growth factors. Compositions can include cells, e.g., at least one of pulp-derived stem cells, progenitor cells, embryonic stem cells, umbilical cord cells, or mesenchymal stem cells. Such cells can be obtained from a subject or from a cell culture (e.g., cells that have migrated from a pulp explant or other tissue explant). In some embodiments, the cells are pulp cells, bone marrow cells, or a combination thereof. In some compositions of the invention, the matrix degrades over time, e.g., after placement in a pulp chamber.

A composition can include chitosan. hi certain embodiments, the composition includes platelet-rich plasma (PRP) or platelet- rich plasma-derived growth factors (e.g., one or more growth factors that are in PRP, or PRP that is prepared to enrich for such growth factors), hi some embodiments, the composition includes at least one of one or more bone morphogenic proteins (BMPs) or dentin powder. hi yet another embodiment of the invention, the matrix is seeded with about 1 x 10⁶ cells/ml, about 2 x 10⁶ cells/ml, or about 3 x 10⁶ cells/ml.

An aspect of the invention relates to a method that includes administering to a subject a physiologically acceptable matrix into the pulp chamber of a tooth. In some embodiments of the method, the physiologically acceptable matrix is seeded with cells. The composition used in the method can include a physiologically acceptable matrix capable of being injected into the pulp chamber of a tooth, hi some embodiments, the pulp chamber is substantially free of native pulp cells. In other embodiments, the pulp chamber comprises native pulp cells. The composition can be, in some cases, inserted apically to the native pulp cells, or the composition can be inserted coronally to the native pulp cells, hi certain embodiments, at least two different compositions are inserted into the pulp chamber. The composition can be administered using a method that includes injection (e.g., of a composition into the pulp chamber of a tooth). In another aspect, the invention relates to a method for treating an individual having a pulp disorder or pulp damage within the pulp chamber of a tooth. The method includes administering a composition that includes a physiologically acceptable matrix into the pulp chamber. In some embodiments, the matrix is seeded with, e.g., pulp cells, embryonic stem cells, umbilical cord-derived cells, or mesenchymal stem cells. In certain embodiments of the method, pulp tissue is removed from the pulp chamber. In other embodiments, pulp tissue is not removed from the pulp chamber. The cells of a composition used in the method can bee derived from the individual (e.g., the individual that is being treated). In other embodiments, the cells of the composition are not derived from the individual being treated. In certain embodiment, following injection of the composition, the pulp chamber is sealed.

The invention also relates to a composition comprising a scaffold of electrospun collagen, electrospun PLGA, degradable polymer, or chitosan mesh, wherein the scaffold comprises at least one antibiotic or growth factor. In some cases, the invention also relates to a method for culturing mesenchymal stem cells or pulp fibroblasts by culturing the cells on a scaffolding composition. In another aspect, the invention relates to a method for culturing primary pulp cells. The method includes seeding the pulp cells that have migrated from a pulp explant in a matrix comprising hydrogel or other matrix as described herein. In certain embodiments, the cells are cultured in a hydrogel and the hydrogel is alginate or chitosan.

Also encompassed by the invention is a kit that includes a physiologically acceptable matrix for seeding with pulp cells and instructions for use. The kit can include, e.g., a medium suitable for maintenance of harvested pulp cells. In certain embodiments, the kit includes sealant suitable for sealing a tooth. In yet other embodiments, the kit includes a chamber for culturing pulp cell cells on a matrix.

Compositions as described herein are also useful in the preparation of a medicament, e.g., for treating a damaged or diseased tooth.

DESCRIPTION OF THE DRAWINGS

Fig. IA is a schematic drawing of an idealized pulp space and enclosing dentin (not to scale) of coronal infected pulp. Vital non-infected pulp is below the dotted line. Infected pulp is above the line. White - dentin, black - dental material, gray - pulp, pulp tissue engineered construct, or regenerated pulp.

Fig. IB is a schematic drawing of a pulpectomized tooth. No pulp remains, and the tooth is restored with dental materials only.

Fig. 1C is a schematic drawing of a tooth undergoing vital tooth therapy as a partially pulpotomized tooth in which only the affected pulp is removed. A pulp-capping agent is placed on top of the pulp wound. Mineralization only occurs below the pulp wound. Fig. ID is a schematic drawing of a tooth undergoing initial level pulp regeneration construct placement for either a pulpectomized or pulpotomized tooth. A pulp regeneration construct is placed coronal to the desired final location of the regenerated pulp.

Fig. IE is a schematic drawing of the final configuration of a tooth with regenerated pulp. Mineralization occurs below the height of the placed regeneration construct.

Fig. 2 A is a reproduction of a photomicrograph of an early stage culture of human pulp cells obtained from explants growing on a tissue culture flask surface and beginning to form an oriented structure.

Fig. 2B is a reproduction of a photomicrograph of human pulp cells obtained from explants growing on a tissue culture flask surface and exhibiting an oriented structure. The culture is more mature than that of Fig. 2A.

Fig. 3 A is a reproduction of a photomicrograph of pulp cells embedded in alginate beads on culture day 3. Original magnification was 1OX.

Fig. 3B is a reproduction of a photomicrograph of pulp cells embedded in a collagen type I gel on culture day 0. Original magnification was 10X.

Fig. 4 is a bar graph depicting results of experiments assaying cell proliferation in which pulp cells grown alone in monolayer were compared to pulp cells grown under co-culture conditions.

Fig. 5 is a bar graph depicting the results of experiments assaying cell proliferation in which pulp cells grown in a chitosan were compared to pulp cells grown in chitosan under co- culture conditions (beads).

Fig. 6 is a bar graph depicting the results of experiments assayed alkaline phosphatase (ALP) in a pulp cell monolayer compared to pulp cells grown under co-culture conditions. ALP activity is normalized and expressed as activity per cell. Fig. 7 is a bar graph depicting the results of experiments assayed ALP in pulp cells grown in chitosan (beads). ALP activity is normalized and expressed as activity per cell.

Fig. 8 is a bar graph depicting the results of cell proliferation assays of human pulp cells cultured on aligned or unaligned nanofϊber mesh.

Fig. 9 is a bar graph depicting ALP activity of human pulp cells cultured on aligned or unaligned nanofϊber mesh.

Fig, 1OA is a reproduction of an SEM photomicrograph of an aligned nanofiber mesh (scaffold) one day after seeding (image x500).

Fig, 1OB is a reproduction of an SEM photomicrograph of an unaligned scaffold one day after seeding (image x500). Fig, 1 OC is a reproduction of an SEM photomicrograph of an aligned scaffold seven days after seeding (image x500). Fig, 1OD is a reproduction of an SEM photomicrograph of an unaligned scaffold seven days after seeding (image x500).

Fig. 1OE is a reproduction of an SEM photomicrograph of an aligned scaffold 14 days after seeding (image x500). Fig, 1 OF is a reproduction of an SEM photomicrograph of an unaligned scaffold 14 days after seeding (image x500).

DETAILED DESCRIPTION

This patent disclosure contains material that is subject to copyright protection. The copyright owner has no objection to the facsimile reproduction by anyone of the patent document or the patent disclosure as it appears in the U.S. Patent and Trademark Office patent file or records, but otherwise reserves any and all copyright rights.

All patent applications, published patent applications, issued and granted patents, texts, and literature references cited in this specification are hereby incorporated herein by reference in their entirety to more fully describe the state of the art to which the present invention pertains.

As various changes can be made in the methods and compositions described herein without departing from the scope and spirit of the invention as described, it is intended that all subject matter contained in this application and claims, shown in the accompanying drawings, or defined in the appended claims be interpreted as illustrative, and not in a limiting sense. The invention provides a functional tissue engineering-based solution to regenerate dental pulp tissue in an affected tooth, e.g., in a tooth that is infected, was infected, or in which the tooth pulp was otherwise damaged, for example by physical trauma. The goal is to restore tooth form and function to approximate those of the pre-affected tooth, for example to preserve nerve innervation of the tooth. The invention provides compositions for use as a functional tissue engineering-based solution to regenerate dental pulp tissue. The invention provides methods to restore the tooth form and function to approximate those of the pre-affected tooth. The clinical significance of such an approach stems from 1) the need for pulp regeneration as reflected by the 24 million root canal therapies performed per year in the U.S., 2) a paradigm shift from the current clinical treatment modality of removing the affected pulp to pulp regeneration, which enables continued dentinal self repair, extended growth of immature teeth, continued ability to fight infection, and the reduction of complications due to prosthetic repair of endodontically treated teeth, 3) the inability of present dental materials to provide permanent microorganism- proof seals whereas pulp and dentin combined provides such a seal, and 4) the lack of viable pulp regeneration grafts. Despite the ability of dental pulp to self-regenerate/repair under specific conditions in vivo, the current major endodontic treatment modality for affected pulp is total or partial removal of affected pulp tissue. Depending on the clinical diagnosis, the treatment goal for teeth with affected pulp is to prevent or treat infection, seal off the pulp space, enable continued root growth in pediatric patients and prevent tooth discoloration. Pulp regeneration is not generally an aim of currently used treatments. The literature on pulp regeneration/repair is small compared to that of dentin regeneration/repair. The focus of treatment is generally on dentin since dentin provides the major form and function of the tooth. Yet, if affected pulp tissue can be regenerated clinically, the regenerated pulp alone could repair/regenerate dentin as it does naturally (Fig. 1).

The invention provides for a composition comprising a tissue engineered construct combining a biocompatible material either with or without a cellular component, which can be used as a solution for pulp regeneration/repair and hence provide for continued normal functioning of the tooth. A functional tissue engineering approach is utilized by the methods of this invention.

The invention encompasses a matrix capable of supporting pulp fibroblast growth and differentiation, while possessing structural and functional properties favorable for implantation. The invention also provides a drug delivery system and a tooth explant organ culture. In some embodiments the invention also provides at least one of an antibiotic, antifungal, antiinflammatory, or angiogenesis drug delivery systems that can be used in conjunction with the pulp regeneration construct.

A composition provided herein generally has specified structural and functional parameters such that the composition can fill the entire pulp space with a dimensionally stable material through which newly generated pulp can grow. This feature of a composition is desirable since voids in the pulp space can increase the chance of infection. The effects cells in the matrix of a composition can be significant since the construct/dentin interface integrity is important. A previous study showed that gel contraction for a collagen/GAG gel was greater in the presence of pulp-cells. ADA (American Dental Association) specification 57 is for endodontic sealants. In current endodontic practice the filled pulp space consists of points (typically thin cones of gutta percha) surrounded by sealant. The present invention relates to the use of a gel, which is not strictly analogous to endodontic sealants, the ADA 57 is used herein as a standard technique to measure gel properties. In another embodiment, the final tissue engineered construct can comprise the addition of point/cone-like material. This more rigid material can be used to deliver drugs or cells. In that case, the gel component of a composition is more analogous to endodontic sealants.

For pulp regeneration to persist, an infection-free and an angiogenic environment is important. An antibiotic delivery system, anti-fungal delivery system, and angiogenic compositions are encompassed by the invention. The invention provides a functional tissue engineering-based solution to regenerate dental pulp tissue. Compositions and methods are provided to restore tooth form and function to approximate those of the pre-affected tooth. The compositions possess structural and functional properties favorable for implantation. In an embodiment, the invention provides a tissue . engineered construct delivered to a damaged tooth combining a biocompatible material either with or without a cellular component that provides a solution for pulp regeneration/repair and consequently, restoring normal function of the tooth.

In general, the invention provides a composition comprising an injectable hydrogel matrix with appropriate viscosity, dimensional stability, microstructure, and mechanical properties for pulp-regeneration.

Matrix Compositions

"Matrix" as used herein comprises a substance suitable for pulp cell culture.

In one aspect of the invention, collagen mixtures are used in a composition that can be used for pulp regeneration and repair. Native pulp tissue is comprised of approximately 55% type

I and 45% type III collagen. One biomimetic composition includes about 55% type I collagen and about 45% type III collagen. Compositions can be optimized based on cellular response and the need to match mechanical properties of the native tissue.

Structural properties that can be evaluated as a function of matrix properties include overall surface and matrix morphology, porosity, and fiber distribution.

Examples of collagen matrices include a type I collagen matrix having a type I collagen concentration of, e.g., about 0.2%-5%, about 0.3%-3.0%, 0.3%, 1.5%, or 3.0% (w/v). Gelation is carried out at a selected pH, e.g., pH 5.0-9.5, 6.0-9.0, 6.0, 7.5, or 9.0.

In some aspects, the matrix is a composite of type I collagen and type III collagen. Non- limiting examples of compositional ratios of type I to type HI collagen include 30%:70%,

55%:45%, and 70%:30% (w/v) and gelation at a selected pH, e.g., pH 5.0-9.5, 6.0-9.0, 6.0, 7.5, or 9.0.

In other aspect, the matrix includes chitosan. Chitosan forms a gel in solutions with a pH above 12, and the gelation occurs at pH of about 9 in 10% amino acid solutions. ^' The matrix can also be an alginate matrix having an alginate concentration of, e.g., 0.5%-

6.0%, 1.0%-5.0%, 1.0%, 3.0%, or 5.0 (w/v). The degree of gelation of an alginate matrix is generally regulated by selecting the CaCl₂ concentration. Non-limiting examples Of CaCl₂ concentrations include concentration from about 25 mM-300 mM, 50 mM-200 mM, 50 mM, 100 mM, and 200 mM. In general, the variations disclosed herein in gel matrix material and gelation conditions are tested in methods for identifying compositions and constructs suitable for use in vital tooth repair, e.g., for administration to the pulp space of a damaged tooth. The matrix can be a chitosan matrix. A chitosan matrix can have a concentration of, e.g., about 1% to about 5%, about 1.5% to about 3%, about 2% to about 4%, about 1% to about 3%, or about 2% to about 3%, e.g., about 2.5%. Methods of preparing a chitosan gel are known in the art, e.g.., using glutaraldehye. Selection of an optimal chitosan matrix can include selecting the degree of deacetylation of the chitosan {e.g., at least about 70%, at least about 80%, at least about

90%, or at least about 95%).

Useful matrices for culturing cells as described herein can also include an electrospun mesh, e.g., made using collagen, chitosan, or alginate. Alternatively, the mesh can be composed of other polymers. Polymer meshes are generally composed of a biodegradable material such as polyøactide-co-glycolide) (PLGA). Methods of making electrospun mesh are known in the art.

In general, the mesh is an aligned mesh, however, the mesh can be only partially aligned or can be unaligned. The mesh can include additional components such as antibiotics and growth factors (Katti et al, J. Biomed. Mater. Res. B. Appl. Biomater., 2004,70:286-296).

Other matrix materials that can be used include hydrogels, MATRIGEL™, gelatin, JELL- O^®, a nanofiber, extracellular matrix, a degradable polymer, a mesh of crosslinked fibers, an artificial or synthetic three-dimensional scaffold material, a polycaprolactone polymer, a polygalactan polymer, a polyanhydride polymer, a mesh fabric, or a combination of any of the foregoing. Methods of preparing such materials are known in the art.

Methods of Increasing Vascularization

Vascularization can be induced by a pulp construct containing a composition disclosed herein by, for example, the incorporation of growth factors such as platelet-derived growth factor (PDGF) and vascular endothelial growth factor (VEGF) into polymer microspheres that are included in a composition, with a matrix such as a collagen gel, chitosan, or alginate. Microspheres containing factors are generally embedded in the gel of the matrix. Other growth factors known in the art such as EGF (epidermal growth factor) and derivatives thereof or a bone morphogenetic factor (BMP) can be used. Platelet-rich plasma and other biological preparations having properties such as promotion of cell proliferation or cell differentiation {e.g., of pulp cells) can be used in a composition. A second mean of encouraging vascularization in a matrix used in a composition is to deposit the above relevant growth factors directly onto a nanofiber mesh using an electrospinning process. This growth factor-containing mesh is embedded inside a hydrogel. This nanofiber mesh serves at least three purposes 1) as the reservoir for angiogenic growth factors, 2) the alignment of the mesh (in the case of an aligned mesh) can guide the formation of blood vessels, and 3) the nanofiber mesh can degrade and make room for vascular ingrowth, cell proliferation, and deposition of extracellular matrix by cells. Similar methods are used for induction of vascularization in a tooth implanted with a construct described herein. In this case, agents that induce vascularization are included in the composition, e.g., in microspheres or using some other delivery method known in the art. Non- limiting examples of vascularizing agents include VEGF or basic fibroblast growth factor (bFGF).

Identification of Suitable Matrix Compositions

Functional properties of a composition that are assayed can include dimensional stability in wet and dry conditions, gel viscosity, as well as elastic modulus. In general, a matrix includes a gel (e.g., a hydrogel such as collagen, alginate, chitosan, MATRIGEL™, gelatin, JELL-O®, polyethylene glycol (PEG), modified PEG, or fibrin), a mesh, a microsphere, and a combination of any of the foregoing. Additional compounds that can be used in a matrix include, without limitation, polylactide-coglycolide (PLGA) mesh, polylactide (PLA) mesh, or polyglycolide (PGA). A mesh can be, without limitation, a cross-linked fiber mesh, a nanofϊber mesh, a mesh fabric, biodegradable polymer mesh, , or a combination of any of the foregoing. Microspheres can be made of any suitable substance, e.g., a biodegradable polymer, a hydrogel, or a combination of any of the foregoing. Also useful for a composition that is a matrix that is a nanofiber, an artificial three-dimensional scaffold material, or a synthetic three-dimensional scaffold material. Additional compounds that can be used as a matrix in the invention include, without limitation, a polycaprolactone polymer, a polygalactan polymer, a polyanhydride polymer.

A matrix useful in the invention conforms to the following matrix selection criteria (termed "optimum criteria" herein). Optimum criteria for a matrix useful for tooth pulp repair can include one or more of the following; 1) low viscosity (<100,000 cp), 2) a setting contraction of less than 10%, 3) a post-setting contraction of less than 1% or less than 0.1% expansion of the matrix after 30 days, and 4) maintenance of structural integrity for up to 30 days, 5) a porous structure that will support cell growth and infiltration as well as nutrient transport, and 6) limited mineralization.

The invention also provides methods for examining the in vitro response (proliferation and differentiation) of pulp fibroblasts to the biodegradable gel matrix (e.g. a matrix containing collagen, chitosan, or alginate), as well as the effect of cell culturing on gel matrix properties (gel contraction and matrix organization). The ability of a matrix to support the growth and differentiation of pulp fibroblasts can be tested as described herein to determine suitability of a matrix for use in tooth repair. There can be a balance between mechanical properties and optimal cellular response (e.g., a highly porous surface may be favorable for cell attachment, but may not have the dimensional stability desired for long term functionality). Thus, certain criteria used for evaluating a matrix are cellular response to an optimized matrix, e.g., cell proliferation and cell differentiation.

A composite matrix of type I and III collagens is one useful matrix as it mimics the native composition of the human pulp, and it can support cell proliferation and differentiation. The presence of type III collagen in a matrix can minimize undesirable mineralization compared to a matrix composed of type I collagen alone.

In some applications, hydrogels such as alginate and chitosan have advantages compared to collagen. For example, hydrogels can be more economical, they can be crosslinked using agents that are not cytotoxic, and, in the case of chitosan, the hydrogel has antibacterial properties that are useful for preventing or ameliorating infection in a tooth when a construct is used for treatment of a damaged tooth.

Chitosan is useful for compositions as described herein. Chitosan is a degradable biopolymer derived from the exoskeleton of crustaceans. The biocompatibility of chitosan is well documented as its anti-bacterial potential. Bacterial infection compromises the pulp vitality and is the primary clinical reason for performing RCT. Accordingly, a use of a matrix such as chitosan that has anti-bacterial properties is useful as a matrix for culturing pulp cells and for compositions for tooth repair.

Pulp fibroblast growth (DNA content) and differentiation (alkaline phosphatase, types I and III collagen production, mineralization, and the expression of osteocalcin and dental sialophosphoprotein) can be examined as a function of matrix type (type I collagen, type I and III collagens, chitosan, or alginate) as well as culture duration (e.g., 1 day, 3 days, 7 days, 14 days, 21 days, or 28 days).

The effect of cell seeding density (e.g., about IxIO⁶, about 2xlO⁶, or about 3xlO⁶ cells/ml) on gel matrix properties (gel contraction and matrix organization) are determined over time (e.g., 1 day, 3 days, 7 days, 14 days, 21 days, or 28 days) to identify conditions that are optimum for compositions containing cells.

Proliferation and differentiation are parameters that can be assessed in both restrained and non-restrained gels to identify an optimum matrix. The optimal gel matrix for pulp tissue engineering is defined as the system that can support pulp fibroblast proliferation and differentiation without causing excessive gel contraction or ectopic mineralization.

The rationale behind the design criteria is several fold. First, the entire empty part of the pulp space should be filled with the tissue engineered construct (matrix plus other components) to prevent infection and permit optimal control of regeneration. To this end, the gel should have a sufficiently low viscosity, so it is injectable and can be used to fill the pulp space. If a gel (e.g., hydrogel) is to be placed in a root canal, a low gel viscosity is beneficial. For example, appropriate visclcities are the same or similar to the viscosities of dental materials such as zinc phosphate and zinc polycarboxylate cements, as well as light consistency silicone and polysulfide impression materials (<100,000cp 2 minutes after mixing) (Vermilyea et al, J. Dent. Res. 56:762- 767, 1977; Reisbick, J. Dent. Res. 52:407-411, 1973; Koran et al, J. Amer. Dent. Assoc. 95:75- 79, 1977). For coronal placement, this requirement is less stringent. Studies injecting light bodied polyvinyl siloxane material into prepared canals of extracted teeth indicate 100,000cp is a maximum viscosity that can be reasonably inserted into the pulp space, e.g., by injection, without excessive force. An intact dentin-gel interface is required in order to avoid microleakage, thus post-setting dimensional stability is significant. According to ADA Spec. No. 57 {American National Standard/American Dental Association Specification No. 57, 2000), the post-setting dimensional change should be less than 1% in contraction and 0.1% in expansion after 30 days.

To test the suitability of a composition, both wet and dry conditions can be investigated.

The integrity of the gel over time is also important. Collagen, alginate, and chitosan can degrade in vivo. This can be a desirable feature of a construct. To test a matrix, 30 days can be used as an initial guide for gel integrity since pulp revascularization in avulsed teeth is reported to be complete within a month.

Compared to many other loose connective tissues, pulp structure has a more open and less fibrous architecture. There is evidence that the pulp itself can regenerate in the presence of nonviable pulp tissue in avulsed teeth. Therefore coronal pulp microstructure is a design mimicked by the compositions of the invention. The coronal pulp structure is more similar to developmental tissue than radicular pulp. Gel structure similar to that of coronal pulp may be more conducive to pulp regeneration. The less fibrous structure of pulp is modeled by varying the percentages of types I and III collagen. The gel mixture with the percentage closest to that found in pulp tissue may be used, such as about 45% type III and about 65% type I collagen. In addition, for pulp regeneration to occur, it is desirable that mineralization be confined mainly to the dentin surface. Some mineralization within the pulp is allowable since many teeth contain mineralized tissue within the pulp but the degree of mineralization should be controlled. The inclusion of type III collagen can aid in the control of non-specific mineralization. In addition, angiogenesis is enhanced in type III collagen versus type I collagen gels.

Unlike some other tissue-engineered constructs, a pulp construct is not required to provide a major role in the overall mechanical function of the tooth. However, during cell culture and in vivo placement, cellular attachment onto the matrix molecules exerts mechanical forces on the gel matrix. These mechanical forces cause significant contraction of the gel. (Brock et al, J. Dent. Res. 81:203-208, 2002) reported that cellular induced contraction also occurs with pulp cells. If the gel is constrained contraction does not occur and mechanical force is exerted to the cells. Since cells are affected by their mechanical environment, growth and differentiation differs on free and restrained gels. Therefore both free and restrained gels are encompassed by the invention. Also, since dimensional stability is desirable within 30 days of implantation, a matrix with higher stiffness can be more desirable. The mechanical properties of the pulp tissue engineered construct may play a role in regeneration.

Estimates of pulp properties for finite element analysis include 0.0 Mpa (Thresher, J. Biomech. 6:443-449, 1973), 0.003 Mpa (Toparli et al, J. Oral Rehab. 26: 157-164, 1999), and

2.0 Mpa (Williams et ah, Biomaterials 5:347-350, 1984). Two other desirable characteristics for pulp regeneration are angiogenesis and an infection free environment. The invention encompasses an optimal matrix capable of supporting pulp fibroblast growth and differentiation, while possessing structural and functional properties favorable for implantation.

Pulp Regeneration

If a pulp regeneration tissue engineered construct is placed coronal to the viable pulp, it is possible to restore both the pulp and dentin closer to the pre-trauma form than with current techniques. With this method, not only would maximal tooth structure be regenerated, but the required traditional restoration would be minimal.

The composition and methods of the invention represent a paradigm shift in treating a traumatized pulp in a tooth. Instead of being removed, pulp tissue is encouraged to form a hard tissue barrier to seal the pulp space for regeneration of pulp to restore and/or replace injured pulp. In some aspects of the invention, stem cells, e.g., stem cells derived from pulp or cranio-facial sources, are used in a construct to promote regeneration of pulp. Regeneration of pulp has advantages over current techniques that are in general use. Regeneration can enable the continued full functioning of the tooth. The ability of the damaged tooth to self-repair, fight infection, and sense stimuli would be re-established. In short, in this proactive approach a tissue engineered construct is placed such that pulp will regenerate through the construct. Two types of pulp constructs can be used. In the first type (termed herein, a "barrier construct"), the construct stimulates only pulp regeneration, and a barrier is placed where initiation of dentin formation is required. In the second type, an interfacial construct is fabricated with the apical portion designed for pulp regeneration and the coronal portion designed for dentin regeneration. This type of construct is termed herein an "interfacial construct." The potential health impacts of a tissue engineered pulp construct over current clinical treatments can include 1) reduce the present treatment-induced removal of sound hard tissue, thereby reducing the need for extensive restorative work, 2) provide a biological seal, thereby decreasing leakage and future ingress of bacteria and other noxious agents, 3) continue and/or improve dentin repair/regeneration, thereby reducing the need for re-treatment due to future pulp assaults, 4) continue and/or improve the pulp host immune system, thereby reducing post- treatment infections and the high costs of re-treatment, and 5) support the growth of immature teeth, thereby reducing the number of tooth fractures. In summary, a pulp construct can reduce the need for re-treatment procedures and increase the life span of the restored teeth.

Pulp Tissue Engineering A rigid resorbable construct such as polygylcolic acid polymer (Bohl et ah, J. Biomaterial

ScL Polym. Ed. 9:749-764, 1998; Bouvier et al, Arch. Oral Biol. 35:301-309, 1990; Mooney e? al, Biotechnol. Prog. 12:865-868, 1996) can be utilized in pulp regeneration (similar to the use of gutta-percha points during root filling) for delivery of cells or cellular mediators. In such constructs a gel-like material is also required to fill the pulp space and thereby prevent infection. This structure that includes the gel-like material with other components such as a rigid resorbable construct, cells, drugs, and growth factors is termed herein a "pulp construct."

One aspect of the invention uses alginate, a hydrogel used in tissue engineering. In another aspect, the gel is a collagen or combination of collagens {e.g., type I and type III collagen). The gel can, in some cases, be chitosan. Optionally, cells can be embedded in the gel during gelation, e.g., to produce a construct that can seed and populate the pulp space and promote pulp regeneration. The cells can be pulp cells, e.g., derived from cultures of pulp cells, or stem cells that can be derived, e.g., from a non-pulp source. Constructs that specifically include cells are referred to herein as "tissue engineered constructs." Phenotypic expression of pulp cells can be manipulated in cell culture (Hao et al, Eur. J. Oral. Sci. 105:318-324, 1997; Couble et al, Calcif. Tissue Int. 66:129-138, 2000; About et al, Exp. Cell Res. 258:33-41, 2000;

About et al, J. Biomed. Mater Res. (Appl. Biomater) 63:418-413, 2002), therefore such manipulation can be utilized in the tissue engineered construct.

In some embodiments, a pulp construct includes a drug delivery system, e.g., to deliver at least one of an antibiotic, anti-fungal agent, angiogenic factor, cell growth factor, nerve growth factor, cell differentiation factor to the pulp space. An anti-inflammatory agent can be included in a construct. Another component of such constructs can be dentin powder. Such agents are known in the art. In some cases, the pulp construct includes chitosan {e.g., as the sole gel component of a pulp construct or as a portion of a pulp construct.

In constructs that include dentin powder, such powder can be obtained from the tooth being treated, e.g. , by means of drilling, obtained from healthy teeth such as wisdom teeth, or from other preparations known in the art.

Delivery systems that can be included in a construct include polymer beads such as those known in the art and used for drug delivery. Non-limiting examples such polymers include poly- alpha-hydroxyester, poly-capralactone, and polyanhydrides. Other drug delivery systems are known in the art and can be adapted for use in the present invention {e.g., U.S. Patent No. 5,308,701, Richardson et al. Nat. Biotechnol 2001, 19:1029-1034). Other drug delivery compositions known in the art can be used in certain constructs.

Clinical Applications In the dental tissue engineering methods described herein, several levels of regeneration are possible, any one of which can improve clinical outcomes. The optimal outcome level is the complete regeneration of pulp structure and function, including odontoblasts and complete innervation and revascularization of a damaged tooth. The next level is the restoration of dentin repair and immune response with incomplete regeneration of structure and function, e.g., incomplete reinnervation. Another outcome is the regeneration of pulp without the dentin repair.

Finally, a vascularized non- pulp tissue can retain a host immune response. Each of the outcome levels represents improved clinical success since they will provide a biological seal, and thereby reduce the post-treatment infection rate. Accordingly, the pulp constructs provided herein can result in an improved or complete biological seal. Non-limiting considerations for clinical application of a pulp construct include: 1) apical foramen, 2) effects on the dentin walls during anti-bacterial irrigation and cleaning of coronal pulp space, 3) attachment of the construct to dentin, 4) cell source, and 5) possible excessive mineralization in the pulp space. These are discussed in additional detail infra.

Dental apical foramen

The apical foramen is a hole or holes at the apex of the root of a tooth through which nerves and blood vessels pass. The apical foramina are large at a young age, especially in the developing tooth, and decrease in size as the tooth grows older. Preservation of the apical foramen is therefore an important consideration in delivering a construct to the pulp space.

Effect of antimicrobial agents

Antimicrobial agents are used to prepare a tooth for receiving a pulp construct. However, some antimicrobial agents are cytotoxic at concentrations in general use and can also adsorb to the dentin. Thus, when such an agent is used to prepare a tooth, the agent may be retained at concentrations that can be toxic to cells within the treated tooth. Examples of such agents include, without limitation, tetracycline, Metronidazole, Ciprofloxacin, Minicyclin and other agents known in the art. Thus, when antimicrobial agents are used as part of a treatment protocol for treatment using a pulp construct, such cytotoxic effects must be minimized. Methods of assaying cytotoxic effects are known in the art. Accordingly, in some cases, an antimicrobial agent or combination of agents that have relatively low cytotoxicity are selected for use with a pulp construct. Alternatively, an antimicrobial agent is selected for its low dentin adsorption properties. Examples of such agents include, without limitation, Ciprofloxacin and Metronidazole. In some methods, an antibiotic local drug delivery system is located within the pulp construct. Examples of such local delivery systems include incorporation of one or more antibiotics into a matrix for extended release, e.g., using polymer microspheres as described herein and as are known in the art.

The use of a pulp construct can include treatment and shaping of dentin walls, which can be beneficial to the success of the tissue engineered construct, for example, because the treatment of dentin walls may cause the release of growth factors that may aid in regeneration.

Attachment of a pulp construct to dentin

An attachment aids in interfacial integrity during possible setting shrinkage, construct contraction by cells, and scaffold degradation. Cells growing on extracellular matrices exert a force on the extracellular matrix molecules. These cellular forces cause the matrix to contract. The contraction is significantly larger than the contraction during polymerization. Agents attaching/securing the gel to dentin can be used to reduce the gel contraction in the presence of cells. Total contraction can also be reduced by the addition of points constructed with polyglycolic acid or its composites (PGA). PGAs are appropriate since pulp cells grow well on PGA (Bohl et al, J. Biomater. ScL Polym. Ed 9:749-764, 1998; Bouvier et al, Arch. Oral Bio. 35:2031-309, 1990; Mooasy et al, Biotechnol. Prog. 12:865-868, 1996). Also, PGAs can be used as a drug delivery system for angiogenesis and antibiotics (Bouhadir et ah, J. Drug Target

9:397-406, 2001; Peters et al, J. Biochem. Mater. Res. 60:668-678, 2002). The points will reduce the volume of gel required and therefore the total overall contraction. Gel contraction can also be controlled by varying gel microstructure, as well as cell culture duration time within the gel before injection.

Cell source

Pulp cells for compositions and methods described herein can be from autogenous or allogenic sources. Alternate sources include expanded cells from the injured tooth, donor pulp cells, stem cells, or host transduced cells. In addition, a construct without cells can be sufficient for pulp regeneration. Other cell types that can be used include embryonic stem cells, mesenchymal cells, umbilical cord-derived cells, stem cells of bone origin, and stem cells of cranio-facial origin. Methods for obtaining stem cells including pulp-derived stem cells, are known in the art (Gronthos et al, J. Dent. Res., 2002, 81 :531-535; Gronthos et al, Proc. Nat. Acad. ScL, 2000, 97:13625-13630).

Mineralization Ectopic mineralization in the tissue-engineered construct can occur and hamper pulp regeneration. Calcified structures occur in teeth — as high as 50% of newly erupted teeth and 90% of older teeth may contain calcified nodules. Although these calcifications are generally non- problematic, their presence indicates that uncontrolled calcification may occur during pulp regeneration. Nonfunctional calcification may be controlled by adjusting the construct material to one that does not favor calcification or by spatially varying the construct materials as well as cellular mediators.

In young teeth damaged by trauma or caries, the coronal and cervical portions of the pulp may be necrotic or severely damaged. Even in these cases, there can be vital pulp tissue remaining in the damaged tooth that is capable of healing in the root canal. The invention thus provides a novel approach to obtain pulp and dentin regeneration in young teeth in which viable pulp is situated apically to the levels where pulp capping and partial pulpotomy would be performed. A restoration of pulp function in the cervical and coronal areas restores dentin formation thereby increasing the hard tissue thickness. TMs prevents cervical fractures, which are common in young teeth that were damaged before cervical hard tissue formation has reached sufficient thickness to reduce the chance of such fractures. A decrease in the restorative needs in such pulp-dentin restored teeth is achieved by the methods and compositions of the invention. In some embodiments of the invention, a pulp construct is combined with other more generally used methods of tooth repair. For example, the area incisally/occlusally located with respect to a placed pulp construct is closed to the oral cavity with composite resin bonded to surrounding enamel. This provides a temporary seal, until a dentin bridge is formed, and fulfills esthetic needs of patient.

Methods The invention provides a functional tissue engineering-based solution to regenerate dental pulp tissue in teeth that are damaged. The methods restore the tooth form of a damaged tooth and function to approximate those of the pre-affected tooth. The compositions comprise a matrix that has structural and functional properties that promote the growth and differentiation of pulp fibroblasts in vitro. Accordingly, the invention relates to methods of identifying compositions that are suitable for promoting the growth and differentiation of pulp fibroblasts in vitro.

Features tested to identify compositions

The invention includes an injectable hydrogel matrix with appropriate viscosity, dimensional stability, microstructure, and mechanical properties for pulp regeneration. In general, the hydrogel matrix is based on one of three types of hydrogel-based materials, collagen type I, combined matrices of collagen types I and III, chitosan, and alginate. The invention encompasses methods to evaluate the suitability of these three types of hydrogel-based materials from structural and functional perspectives.

The methods encompass measuring the structural and functional properties of the resultant matrices generated by controlling the parameters listed in Table 1. Specific examples of such structural and functional properties include morphology, porosity, fiber organization, dimensional stability, viscosity, and mechanical properties under confined compression. Table 2 provides additional detail as to the features of these structural and functional properties that can be

10 used to indicate the suitability of a cell or cells cultured by a selected culture method.

Construct and Matrix Fabrication

Matrices are compared for their suitability as matrices for growth of pulp fibroblasts ,

15 e.g. , for promoting cell proliferation and/or differentiation, and for implantation purposes. In some cases, a construct includes a gel but does not include a rigid component. One such construct includes alginate, a biocompatible, degradable biomaterial widely used in soft tissue engineering. The matrix can be is compared in surface, structural, and mechanical properties with other matrices containing, e.g., type I collagen, as well as composites of types I & HI collagen matrices.

20 These collagen types are selected based on features of native pulp tissue, which is comprised of approximately 55% type I collagen and 45% type IH collagen (van Ameronsen et ah, Arch. Oral Biol. 28:339-345, 1983). For example, different alginate gels can be fabricated by varying the alginate concentration {e.g., 1%, 3%, or 5% (w/v)) and CaCl₂ concentration (for example, 50 mM, 100 mM, or 20OmM CaCl₂). Alginate gels are formed by mixing alginate solution (Sigma, St. Louis, MO) with a known concentration of CaCl₂ solution in a rectangular mold. After the gel has set (about 15 minutes), discs of pre-determined size are corked from the rectangular gel and used for culturing cells. Both type I (0.3 mg/ml, 1.5 mg/ml, and 3mg/ml final concentration in the gel) and type I and type III composite collagen gels are also useful. The polymerization variables for assays used to select suitable constructs are the collagen solution concentrations and gelation pH (pH 6, pH 7.5, pH 9). Briefly, in these experiments, collagen (Invitrogen) is first dissolved in 0.01 M HCl, and gelation occurs after neutralizing the collagen solutions in 10x PBS and incubation at 37⁰C. The type I and type HI composite collagen gels are manufactured by combining type I and type IE collagen in various ratios (for example, LIII wt% ratios of 30:70, 55:45, and 70:30), and gelation following the method described supra.

Chitosan gels are fabricated by cross-linking chitosan using methods known in the art. In general, gels for a pulp construct can be made in the presence of other components of the construct such as beads containing one or more drugs, growth factors, or differentiation factors.

) Construct and Matrix Characterization — Structural and Functional Properties

For a gel matrix, surface morphology and overall matrix organization is determined as a function of the variables listed in Table 1. The surface morphology, matrix organization, fibril number and diameters are examined via scanning electron microscopy (SEM, JEOL 5600LD, 5keV), confocal microscopy, and phase contrast microscopy. Organization in the gel interiors or cross sections will be also be, examined by SEM. Matrix porosity is determined via quantitative image analysis of the SEM and light microscopy images using the Zeiss Axiovision modular image analysis package (Axiovision 3.1, Zeiss). The fibril density, diameter, and length of the type I and type I and HI collagen gels is examined via confocal microscopy (Roeder et al, J. Biomed. Eng. 124:214-222, 2002) and quantitative image analysis. Other methods known in the art for visualizing a matrix and assessing structural and functional properties can be used.

Dimensional stability

Dimensional stability is the ability of a substance (e.g., matrix) to resist changes caused by environmental factors. In the case of a matrix as used herein, dimensional stability relates to dimensional changes following gelation. Dimensional stability is measured following the methods described in the American Dental Association (ADA) Specifications No. 57; the methods described in the specifications can be applied to the matrices described herein as standardized techniques to measure gel properties. Gel viscosity can also be measured, e.g., using an Ubbelohde viscometer (Cannon Instrument Co., State College, PA). Kinematic viscosity is calculated by multiplying the efflux time by the viscometer constant. Viscosities are measured for, e.g., 0 minutes, 5 minutes, and 15 minutes.

Functional properties

Functional properties of the gels are also examined in relation to matrix compositional and fabrication parameters. Although the mechanical properties of pulp tissue have not been well defined, and there are limited publications on the effects of mechanical stress on pulp and dentin regeneration, the mechanical properties of the hydrogel are directly related to its dimensional stability. Therefore, gels are tested under conditions of confined compression to determine their mechanical properties. Specifically, the mechanical properties of the gels are determined in confined compression using the method of Mauk and Ateshian et al. (Mauck et al, J. Biomech. Eng. 122:252-260, 2000). In addition, the modulus and compressive strength of the hydrogel matrices are compared to human pulp tissue tested under identical conditions to develop a matrix that mimics the properties of human pulp. For example, coronal pulp tissue is frozen and later shaped into approximately 3 mm wide cubes, and the mechanical properties of the coronal pulp tissues are measured along the long axis of the tooth as well as the two perpendicular axes. A confined compression test will utilize a rigid-porous permeable sintered steel indenter. The testing system consists of a computer controlled stepper micrometer displacement actuator, a linear variable displacement transducer (LVDT) to measure strain, and a load cell to measure stress. Before testing, free swelling disk thickness is measured with a current sensing micrometer. With a ramp speed of 1 μm/s, deformations on the order of 10%, 20%, and 30% are applied. For each deformation, stress-relaxation curves (force vs. time) are recorded utilizing a 10 g load cell. The equilibrium modulus is determined for each applied deformation, based on the equilibrium force determined from stress-relaxation curves.

In vitro Testing of Gel Matrices To further test the properties of a gel and to identify a gel that is useful for culturing pulp fibroblasts, in vitro responses of pulp fibroblasts are assayed in a test gel, e.g., cell proliferation and differentiation of cells grown in contact with the matrix are tested. In addition, the effect of cell culture on the properties of the gel matrix are assayed (e.g., gel contraction).

The biocompatibility, and the potential of the a matrix to support the growth and differentiation of pulp fibroblasts can be examined, e.g., to identify a matrix suitable for culturing pulp fibroblasts and to serve as a matrix for transplanted pulp fibroblasts. In some embodiments, a composite matrix of type I and type III collagen is used as it closely mimics the native composition of the human pulp and can support cell proliferation and differentiation. Table 3 provides non-limiting examples of types of matrices, and the seeding density and culture duration that can be used to assess the ability of a matrix to promote cell proliferation and differentiation.

The effect of the above two parameters on the proliferation and differentiation of pulp fibroblasts can measured as detailed in Table 4

Table 4

Other methods known in the art can be used to assay markers of cell proliferation and pulp fibroblast differentiation.

Establishment of Human Pulp Fibroblast Primary Cultures

In some cases, the pulp constructs and methods for use of such constructs include the use of pulp fibroblasts. Accordingly, methods of culturing such fibroblasts for use for pulp constructs and for testing certain features of a pulp construct are encompassed by the present inventions.

To culture human pulp fibroblasts, non-carious premolars and third molars from healthy individuals are collected, e.g., from surgical waste. Tooth surfaces are washed with 70% ethanol and the pulp is extracted after cracking the teeth. The pulp is washed five times with wash solution (Dulbecco's Modified Eagles Medium (DMEM), supplemented with 2% penicillin (10,000 IU) - streptomycin (lOmg/ml) solution, and 5.0 μg/ml of amphotericin B (Sigma, St. ' Louis, MO)). The pulp is cut into cubes with approximately 2 mm edges with a sterile surgical blade and placed inside a T-75 flask with a few drops (about 3-5 ml) of the above wash solution.

To insure that the pulp pieces attach to the bottom of the flask, the flask is then placed in an incubator for 45 minutes. After attachment, a minimal amount of explant media (wash solution + 10% FBS) is added to the flasks. Cellular outgrowth is monitored after 10 days of incubation. At least about 50 cells is considered sufficient outgrowth. If there is sufficient outgrowth, explant medium is added to bring the medium volume to approximately 13 ml. Once colonies form around the explants (a few hundred cells; about day 14) the medium composition is changed from the above explant medium to an explant medium containing wife half the concentration of pen/strep and antifungal agent. The medium is then changed every other day with 13 ml of medium. The time required to reach confluency depends on the number of attached pulp explants in the T-75 flask, but generally occurs about six weeks after tooth extraction.

Cell Culture and Seeding on Optimized Hvdrogel Constructs

Primary human pulp fibroblasts are cultured in DMEM + 10% FBS and 1% penicillin (10,000 IU) — streptomycin (10 mg/ml) solution, and 2.5 μg/ml of Amphotericin B, at 37⁰C and 5% CO₂ until confluence. First passage cells are embedded in the hydrogels and cell growth and differentiation is examined as a function of gel type, seeding density, and culturing duration.

To embed the cells in collagen, cell suspensions are added to collagen solutions after neutralization, but before polymerization. Briefly, 1.5 ml of pulp cell suspension in DMEM is mixed on ice with 5.0 ml of 3.1 mg/ml collagen solution (Vitrogen), 0.5 ml HEPES (25 mM), and 0.5ml DMEM. Examples of final cell seeding densities and collagen concentrations are listed in Tables 2a and Ia respectively. The collagen/cell solution is then poured into a square mold inside a petri dish. The dish is incubated for 2 hours at 37⁰C to allow polymerization to occur. The collagen mold system utilized was developed by Holmes et al. (Biomech, Model. Mechanobiol. 2002, 1 :59-67). Briefly, a 4 cm x 4 cm square mold (inner dimensions) is housed in a 100 mm x 15 mm petri dish. Porous polyethylene bars (2 cm x 5 mm x 3 mm) are placed on the edges of the inner mold. Sutures are placed around the bars. The collagen mixture is poured into mold. After polymerization the mold is removed leaving a square collagen gel. For restrained gels, the sutures are taped to the petri dish thereby preventing contraction of the gel. For unrestrained gels the sutures remain unattached.

Cells are embedded in alginate by first combining fibroblasts in alginate solution. The gelation procedure is described herein.

Characterization of Human Pulp Fibroblast Response to Optimized Hvdrogel Constructs Cellular attachment and growth morphology

To assay the effect of a hydrogel composition on human pulp fibroblasts, cellular attachment and growth morphology are be examined. For example, the characteristics of attachment and growth morphology can be assayed using histological staining and scanning electron microscopy (SEM, JEOL 5600LD, 5keV). Non-adherent cells are removed by washing cultured pulp fibroblasts at selected time points, e.g., the samples are washed three times with PBS to remove non-adherent cells.

For cytochemical staining, samples are fixed in 4% paraformaldehyde, dehydrated in ethanol and embedded in paraffin. The samples are then sectioned and stained with hematoxyline and eosin using methods known in the art. For SEM analysis, the samples are first dehydrated using an ethanol drying series, and then left to dry in Freon overnight in a chemical hood. Prior to imaging, the samples are coated with carbon to eliminate charging effects. Cell proliferation is determined using the PicoGreen® dsDNA quantitation assay (Molecular Probes, Carlsbad, CA) where fluorescence intensity is correlated with DNA concentration. In general, it is desirable that cultured pulp cells have features of proliferating cells and the capacity to express proteins indicative of further development to become functional pulp cells.

Phenotype assays Assays can be performed to ascertain the phenotype of the cultured pulp fibroblasts. In general, a cultured pulp fibroblast that is suitable for use in the invention {i.e., for implantation) exhibits at least one of the following, alkaline phosphatase synthesis, osteocalcin production, dentin sialophosphoprotein, or both types I and III collagen synthesis. Immunofluorescent staining can be used to qualitatively examine the expression of these proteins by these cells following the methods of Gronthos et al. (Gronthas et al, PNAS 97: 13625-13630, 2000; Gronthas et al, J. Dent. Res. 81 :531-535, 2002). Alkaline phosphatase expression is quantified using a colorimetric assay. In such assays, a sample is incubated at 37⁰C for 30 minutes in 0.1 M Na₂CO₃ buffer containing 2 mM MgCl₂ with disodium p-nitrophenyl phosphate (PNP-PO₄) as the substrate. Standard solutions are prepared by serial dilutions of 0.5 mM p-nitrophenol (pNP) in Na₂CO₃ buffer. Enzymatic activity is expressed as the total nmoles of pNP produced per minute per total cell number. Absorbance is measured at 415 nm using a Spectrofluor reader (Tecan). The synthesis of type I and III collagen by the human osteoblast-like cells is quantified using a modified ELISA assay used by Lu et al (Barthel et al, J. Endod. 26:525-528, 2000). The expression of osteocalcin and dentin sialophosphoprotein (DSPP) are detected by reverse transcription followed by polymerase chain reaction (RT-PCR). For this purpose, cells are released from alginate by standard procedures using sodium citrate, and from collagen gels using collagenase digestion. Cells can be released from chitosan using methods known in the art, e.g., chitinase digestion. Total RNA is isolated using Rneasy® kit (Qiagen, Valencia, CA). First strand cDNA is synthesized using Superscript (Invitrogen). PCR is performed using the following primer sets: osteocalcin, sense 5'-CATGAGAGCCCTCACA-S' (SEQ ID NO:1) and antisense 5'-AGAGCGACACCCTAGAC-S' (SEQ ID NO:2); DSPP, sense 5'- GGCAGTGACTCAAAAGGAGC-S' (SEQ ID NO:3) and antisense 5'-

TCATATTTGGCAGGTTTTTCT-S' (SEQ ID NO:4) (Gronthos et al, J. Dent. Res. 81:531-535, 2002; Gronthos et al, Proc. Nat. Acad. Sd. USA 97: 13625-13630, 2000). PCR is performed for 35 cycles at annealing temperature of 56°C. PCR products are analyzed using 1.5% agarose gel electrophoresis and visualized by staining with ethidium bromide.

In some cases, the formation of a mineralized matrix by the cultured pulp fibroblasts is determined. Although pulp fibroblasts do not generally exhibit mineralization under physiological conditions, the ectopic formation of mineralized nodules by these cultures in the optimized matrices is examined by SEM/EDXA, and, if necessary, the specific Ca/P ratio IS calculated based on a hydroxyapatite standard. Mineralization can be further confirmed using

Alizarin Red S (ALZ) staining specific for calcium. The samples are washed in double distilled H₂O, and incubated in 40 mM Alizarin red solution for 10 minutes. After additional washes, the scaffolds are incubated in 10% cetyl pyridinium chloride for 15 minutes to solublize reacted ALZ. In this assay, serial dilutions of 1 M CaCl₂ are used as standards. ALZ concentration per cell is calculated as molar equivalent CaCl₂ divided by the average cell number. Absorbance is measured at 570 nm using a Tecan Spectrofluor system (Tecan, Durham, NC).

Other methods known in the art can be used to assay the marker proteins or RNAs. The expression of these proteins in cultured pulp fibroblasts indicates that such cells are suitable for use in implantation, e.g., to repair a tooth. In general, suitable conditions for culturing a pulp cell are those in which at least 80%, e.g., at least 90% of cells express one or more pulp cell markers.

Transplantation and Treatment of a Damaged Tooth

To administer a construct to the pulp space, the entire treatment procedure is performed under aseptic conditions with the use of a rubber dam (to keep saliva, etc. away from the treatment site), sterilization of the field of work, and the use of sterile instruments. Treatment includes at least the following; 1. Mechanical removal of caries that may be present; 2. mechanical removal of infected and damaged pulp tissue that may be present; 3. irrigation of the treatment site with an antibacterial solution; 4. treatment of dentin surfaces with e.g., EDTA; 5. placement of a construct; and 6. sealing of the treatment area.

Animal Models

Constructs can be tested using animal models for tooth damage, e.g., canine models (e.g., Skoglund and Hasselgren, Oral Surg. Oral Med. Oral Pathol. (1992) 74:789-95; Skoghxnd, Int. J. OralSurg. (1983) 12:31-38; and Hasselgren et al, (1977) Oral Surg. 44:106-112). Methods used for implantation of a construct are described herein and methods are known in the art. Data Analysis

For data analysis, primarily two-way analysis of variance (ANOVA) is performed to determine any statistically significant relationship between factors examined in the proposed experiments. Once a significance difference is predicted by ANOVA, the Tukey-Kramer significance test is used to compare between the group means. Statistical significance is tested at p<0.05. Statistical analysis can be performed using the Sigma Stat statistic software (SPSS) or other suitable software.

The specific variables that are measured include porosity, dimensional stability, viscosity, mechanical properties, as well as fiber diameter and length. The factors are percent gel material in the composition, e.g., the percent of collagen, alginate, or chitosan in solution and either CaCl₂ concentration for alginate or pH for collagens. The effect of gel composition can also be analyzed.

The specific variables to be measured generally include light and SEM image analysis of cells, alkaline phosphatase expression, DNA synthesis, osteocalcin expression, and dentin sialophospho protein expression. The factors are culture duration and seeding density.

Pulp Regeneration

If a pulp regeneration tissue engineered construct is placed coronal to the pre-trauma pulpodentin interface, it is possible to restore both the dentin and the pulp closer to the pre-trauma form than with current techniques. With this method, not only is maximal tooth structure be regenerated but the required traditional restoration is minimal (see Figs. 1A-1E). Although some dental material may be required, regeneration is maximized and tooth life expectancy increased. The restored tooth is closer to its original form. In some cases, such methods can decrease the clinical technical complexity.

A pulp regeneration tissue engineered construct includes at least a gel matrix (e.g., a collagen matrix, or a hydrogel such as an algenate matrix or a chitosan matrix), and optionally, pulp fibroblasts, m addition, the construct can include one or more of an antibiotic, an antifungal agent, or a growth factor (including one or more factors that promote cell proliferation and/or differentiation). The invention represents a paradigm shift in treating traumatized pulp. Instead of being removed, pulp tissue is induced to regenerate. In some cases, the regenerating pulp can form a hard tissue barrier in order to seal the pulp space. Regeneration of pulp has several advantages over current techniques for treating a damaged tooth. Regeneration enables the continued full functioning of the tooth. The ability of a damaged tooth to self-repair, fight infection, and sense stimuli can re-established using pulp regeneration methods as described herein. In short, in this proactive approach, a tissue engineered construct through which pulp can regenerate is placed within a tooth.

Two types of constructs are provided herein. In the first, the construct stimulates only pulp regeneration and a barrier is placed where initiation of dentin formation is required. This type of construct is referred to herein as a "simple pulp regeneration construct." In the second type, an interfacial construct is fabricated with the apical portion designed for pulp regeneration and the coronal portion designed for dentin regeneration. This type of construct is referred to herein as an "interfacial construct."

Possible Health Impact of Tissue Engineered Aided Pulp Regeneration

The potential positive health impacts of a tissue engineered pulp construct over current clinical treatments include 1) reducuction in the present treatment-induced removal of sound hard tissue, thereby reducing the need for extensive restorative dental work, 2) provides a biological seal decreasing leakage and future ingress of bacteria and other noxious agents, 3) continue and/or improve dentin repair/regeneration, thereby reducing the need for retreatment due to future pulp assaults, 4) continue and/or improve pulp host immune system reducing post-treatment infections and the high costs of retreatment, 5) support the growth of immature teeth thereby reducing the number of tooth fractures. Therefore, successful utilization of such a construct can reduce the need for retreatment procedures and increase the life span of the restored teeth.

Design of Tissue Engineering of Pulp Constructs

The entire pulp space must be filled with the tissue engineered construct for optimal control of regeneration and to prevent infection. To this end, the gel should have sufficient viscosity and volume to be injected into the entire space. Dimensional stability before degradation is desired. Factors affecting stability are setting contraction, contraction due to applied forces by cells, thermal cycling contraction, and premature degradation. The methods described in ADA Spec. No. 57 can be used as a standard to measure dimensional stability. An intact dentin construct interface is required to avoid microleakage. A pulp regeneration time of thirty days can be used as an initial guide for construct degradation. Varying composition, molecular weight, and microstructure can control these properties. The presence of cells can affect dimensional stability and degradation. Therefore these two properties must be remeasured in the presence of cells. It is unclear to what extent construct mechanical properties may play a role in pulp regeneration. Unlike some other tissue engineered constructs, the pulp construct is not required to provide mechanical support in order for the tooth to function. However, mechanical properties may play a role in the pulp tissue construct's success. For example, the presence of pulp cells can cause significant additional contraction of a three dimensional gel. Since dimensional stability is a requirement, a stiffer gel than that used in that particular study may be needed. In addition, the mechanical environment affects the behavior of many cell types. Therefore, the mechanical properties of the pulp tissue engineered construct can play a role in regeneration.

Clinical Application

In any dental tissue engineering application, it is possible to have several levels of regeneration and still improve clinical outcomes. The optimal level is the complete regeneration of pulp structure and function, including odontoblasts and complete innervation and revascularization. The next level is the restoration of dentin repair and immune response but an incomplete regeneration of structure and function. A third possibility is the regeneration of pulp without the ability to repair dentin. The final possibility is the regeneration of a vascularized non- pulp tissue providing both a biological seal against microleakage, as well as retaining a host immune response. Thus, the methods of the invention will improve clinical success since they will provide a biological seal, thereby reducing the post-treatment infection rate.

Possible challenges to clinical application include: 1) reduced apical foramen size in mature teeth, 2) effects of traditional endodontic treatment on the dentin walls during antibacterial irrigation and cleaning and shaping with root canal instruments, and 3) source of cells. If the apical foramen width is narrow, revascularization does not occur. For pulpectomy treated teeth with narrow apecies the apex may have to be widened. Although such widening is possible since canals are shaped during endodontic treatment, widening the apex without damaging the periradicular bone and ligament will present technological issues.

Treatment of the dentin walls with currently used antimicrobial agents can be evaluated for cytotoxicity since such agents can be cytotoxic at the commonly used concentrations. These agents may initially be adsorbed into the dentin and released with time. Antimicrobial agents that are less cytotoxic or that do not readily adsorb into the dentin can be utilized. Such agents can be identified using the culture methods described herein and testing an agent for efficacy and cytotoxicity using methods known in the art. Also the inclusion of an antibiotic local drug delivery can reduce the level of antimicrobial required. The treatment and shaping of dentin walls may actually be beneficial to the success of the tissue engineered construct. Finally, the treatment of dentinal walls may cause the release of growth factors that may aid in regeneration. Also, dentin treatment can allow the use of dentin bonding agents to increase the strength of the interfacial bond between construct and dentin. A stronger bond would aid in interfacial integrity during possible setting shrinkage, oral thermal cycling, construct contraction by cells, and scaffold degradation. A large number of cells may be needed in the repair of critical size defects such as after pulpectomy. At the present time the use of pulp cells from autogenous or allogenic sources are highly impractical. Alternate sources to be considered include expanded cells from the injured tooth, donor pulp cells, stems cells, or host transduced cells. In addition, ectopic mineralization in the tissue engineered construct may occur and disrupt the path to pulp regeneration. Calcified structures are fairly common, as high as 50% of newly erupted teeth and 90% of older teeth may contain calcified nodules 64. Although these calcifications are normally non-problematic, their presence indicates uncontrolled calcification may occur during pulp regeneration. Nonfunctional calcification may be controlled by adjusting the construct material to one that does not favor calcification or by spatially varying the construct materials as well as cellular mediators.

In general, after a pulp construct is placed within the pulp space of a tooth, it is necessary to provide at least a temporary sealant. Such sealants are known in the art, e.g. , a composite resin that can be bonded to surrounding tooth structure. The features of such a sealant include, when set, biocompatibility and bonding properties that are maintained for a sufficient amount of time for the pulp to recover and regenerate a seal.

The invention encompasses an injectable hydrogel matrix with appropriate viscosity, dimensional stability, microstructure, and mechanical properties for pulp regeneration. An evaluation the suitability of three types of hydrogel-based materials (chitosan, alginate, collagen type I, and a combined matrix of collagen type I/Ill) from a structural and functional perspective is provided.

Compositions as described herein can be provided in kits suitable for preparation of a construct and treatment of a damaged tooth by a practitioner. Kits can be for treating specific conditions, e.g., for treating a tooth with remaining pulp apically, a tooth without living pulp cells (where the kit has to provide e.g. stem cells). A kit may also contain components needed for a practitioner to collect a pulp cell sample from a patient (e.g. , by providing a culture container and medium with suitable components such as antibiotics), which is sent to a laboratory for preparation of a construct.

Construct Fabrication and Optimization Alginate gels can be fabricated by manipulating two parameters, alginate concentration

(for example, 1%, 3%, or 5% (w/v)) and CaCl₂ concentration (5%, 10%, 20% (w/v) CaCl₂).

Alginate gels by mixing alginate solution (Sigma) with CaCl₂ solution. The alginate solution can be dropped into a stirred CaCl₂ solution. Spherical beads are formed during gelation. Beads are removed from solution after 60 minutes and washed in distilled water. Several different collagen gels can be fabricated, for example, gels containing a single type of collagen, as well collagen mixtures with type Ltype III ratios of 30:70, 55:45, and 70:30. The polymerization variables are the collagen solution concentrations (0.3 mg/ml, 1.5 mg/ml, 3mg/ml final concentration in the gel) and the pH (6, 7.5, 9). Collagen is dissolved in 0.01M HCl. Gelation occurs by neutralizing the collagen solutions in 1OX PBS and placing in a 37⁰C incubator. Total phosphate ionic strength is held constant at 0.14 M. Chitosan gels can be formulated using methods known in the art and illustrated in the

Examples {infra).

Gel microstructure and porosity is determined via image analysis. For alginate the beads are cryo-fractured and the surface and interior examined with SEM. For the collagen gels the fibril density, diameter, and length is determined via confocal microscopy (Rungvechvottivittaya et al, Arch Oral Biol. 43:701-710, 1998), although SEM can also be used. Chitosan gels can be examined using confocal microscopy or SEM. Other suitable methods for examining gel morphology and known in the art can also be used for such examinations.

Dimensional stability is analyzed using ADA Spec. No. 57 as a guide. Measurements are made in both dry and wet conditions at 37⁰C for 1 day, 2 days, 7 days, 14 days, 21 days, and 28 days. For wet conditions the gel is placed on a Millipore filter. This filter is placed on a cylindrical support and put into a petri dish. DMEM is added to the dish up to the height of the filter paper.

The Ubbelohde viscometer is used to measure kinematic viscosity for all gels. Kinematic viscosity is calculated by multiplying the efflux time by the viscometer constant. Viscosities are measured for various times, e.g., 0 minutes, 5 minutes, 2 hours, 10 hours, and 3 days.

Gels are tested in confined compression to determine their mechanical properties (Ranley et al., J. Dent. 28:153-161, 2000). The modulus is acquired from the equilibrium force determined from stress-relaxation curves. Confined compression test utilizes a rigid-porous permeable sintered steel indenter. The unconfined compression test utilizes a rigid-impermeable glass loading platens. Before testing free swelling disk thickness is measured with a current sensing micrometer. The custom designed testing system consists of a computer controlled stepper micrometer displacement actuator, an LVDT to measure strain, and a load cell to measure stress. With a ramp speed of lμm/s deformations of, e.g., 10 %, 20 %, and 30% are applied. For each deformation stress-relaxation curves (force versus time) were recorded utilizing a 10 g load cell. The equilibrium modulus was determined for each applied deformation.

The invention provides methods to modulate the proliferation and differentiation of pulp fibroblasts in the biodegradable gel matrix. The structural and functional similarities to native pulp tissue, the ability of the experimental matrices to support the growth and differentiation of pulp fibroblasts are examined. The composite matrix of type I collagen and type III collagen is one useful matrix because it mimics the native composition of the human pulp and supports cell proliferation and differentiation. EXAMPLES

The invention is further illustrated by the following examples. The examples are provided for illustrative purposes only. They are not to be construed as limiting the scope or content of the invention in any way.

Example 1: Establishment of Pulp Fibroblast Cultures

A protocol for establishing human pulp cells from explant outgrowth has been developed and is described herein. For preliminary purposes, the outgrowth of porcine pulp tissue was investigated. A suitable porcine model for developing pulp cultures is useful for preliminary trials and for comparison to human pulp tissue.

The methods used were as described supra for explanting human pulp tissue and culturing such tissue for outgrowth and subsequent culturing of cells. Porcine pulp fibroblasts were observed to grow faster than human pulp cells and their attachment morphology was slightly different from those of human origin. Initial and continued explant attachment to the culture flask surface was critical for outgrowth to occur. Contrary to other soft tissues, pulp explants that floated in culture medium did not produce cells. Explant pieces that became dislodged were removed from the flask and reattached to the bottom of another flask. Such reattached explants were able to produce cells. It was observed that outgrowth from porcine explants began as early as 3 days and as late as 12 days. Colonies that were visible by eye grew around the explants.

Over time, colonies also formed at non-explant sites. The cell morphology varied with culture duration from round, to stellar, to elongated (Fig. 2 A and Fig. 2B). Elongated cells eventually formed an oriented structure. After significant outgrowth occurred, i.e., a few hundred cells, the time for the culture to reach confluency in a T-75 flask varied from three to six weeks. Time from tooth pulp extraction to obtaining a flask of confluent cells ranged from about one to two months.

Trypsinizing the explant outgrowth cells yielded approximately IxIO⁷ cells per flask (SD=9xlO⁶).

Example 2: Collagen and Alginate Gel Fabrication and Cell Embedding

Human pulp cells were embedded and grown in type I collagen gel for 6 weeks as described herein. To prepare a matrix containing pulp cells and collagen, 1.5ml of cell suspension

(3.IxIO⁶ cells/ml) in DMEM was mixed on ice with 5.0 ml of 3.1 mg/ml collagen solution (Vitrogen, Palo Alto, CA), 0.5 ml HEPES (25 mM) and 0.5 ml DMEM to a final concentration of 2.0 mg/ml of collagen in the collagen-cell solution. Collagen-cell solution was poured into the wells of a 6 well culture plate. The plate was incubated for 2 hours at 37⁰C. After polymerization, the culture media was added. Media was changed every two days. After 14 days these unrestrained gels containing cells exhibited noticeable contraction and became detached from the well surfaces. After 40 days, the free-floating collagen gel containing cells contracted 40% in diameter. Controls without cells exhibited no noticeable contraction. Cell morphology in collagen gel changed with time from an initial rounded shape to an elongated shape similar to cells grown on the culture flasks. In addition, ALP was observed to be increased in cells cultured in the type I collagen matrix.

Human pulp fibroblasts were also embedded and grown in alginate gel. Cells (2.8xlO⁶ cells/ml) were mixed with either a 1.0 % wt/vol or a 3.0 % wt/vol alginate solution (Sigma). The cell-alginate solution was dropped into a stirred 100 mM CaCl₂ solution through a 21 gauge needle. Beads were removed from solution after 15 minutes and washed in PBS. The cell embedded in alginate was placed in the culture media solution and stored at 37⁰C incubator with

5% CO₂. Culture medium was changed every two days.

It was observed that the cells remained viable in the alginate gel and were homogenously distributed through out the gel (Fig. 3A). In contrast to collagen gels (Fig. 3B), there was no change in cellular morphology in alginate with time.

Example 3: Mechanical Properties of Porcine Pulp Tissue

An unconfmed compression testing apparatus (of a type that can be used for cartilage explant testing) was used (Mauck et al., J. Biomech. EngΛ22:252-260, 2000) to test hydrogel mechanical properties. Porcine tissue was used as a testing tissue as it is relatively readily available compared to human pulp tissue. For the testing, the sample was cut into a 4x4x2mm section, with the 2 mm axis in the buccal-lingual direction. A tare load of 2 g was applied. With a ramp speed of 1 μm/s, deformations of 10 % and 20% were applied. For each deformation stress-relaxation curves (force vs time) were recorded utilizing a 50 g load cell. The equilibrium modulus was determined for each applied deformation. The measured modulus was calculated to be 1.3 kPa. Previous estimates of pulp properties in the dental finite element literature include 0

Mpa (megapascals) (Thresher, J. Biomech. 6:443-449, 1973), 0.003 (Toparli et al, J. Oral Rehab. 26:157-164, 1999), and 3 Mpa (Williams et al, Biomaterials 5:347-350, 1984). An increased sensitivity may be gained with a 10 g load cell instead of the 50 g load cell utilized as described herein. With a 10 g load cell, a lower smaller tare load and smaller specimen size can be used. The invention comprises established primary human pulp fibroblast cultures and methods for culturing; methods for embedding pulp cells in both type I collagen and alginate hydrogel matrices; and methods for determining the mechanical properties of human pulp.

Example 4: Establishment of Human Pulp Cell Cultures From Pulp Explants To obtain cells suitable for testing matrices and constructs, and for transplantation using such matrices and constructs, pulp cell cultures are established. In one example of such culture preparation, non-carious premolars and third molars from healthy individuals were collected, e.g., from surgical waste. Tooth surfaces were washed with 70% ethanol. Teeth were cracked to extract the pulp tissue. Pulp was removed from the cracked tooth under sterile conditions.

The pulp is washed five times in DMEM supplemented with 2% penicillin (10,000 IU) - streptomycin (10 mg/ml) solution, and 5.0 mg/ml of amphitericin B (Sigma, St. Louis, MO). The pulp was cut into pieces with a sterile surgical blade. Pulp pieces generally have sides approximately 2 mm in length. Pulp pieces were removed from the petri dish and placed inside a T-75 flask with only a few drops of wash solution. To insure that the pulp pieces attach to the bottom of the flask, the flask was then placed in an incubator for 45 minutes. If pieces do not attach, some wash solution may be removed or the solution spread by rocking the flask and placing it back in the incubator for another 15 minutes. After attachment a minimal amount of "explant medium" (wash medium with 10% FBS) was added to the flasks. The flasks were then incubated for about 10 days.

After the incubation period, the flasks were gently removed from the incubator. If there was sufficient outgrowth (approximately 50 cells), explant medium was added to bring the medium volume to approximately 13 ml. During this time if any explant pieces were floating they can be removed, for example, using a sterile pipette, and placed into a T-25 flask for reattachment. Once colonies formed around the explants (a few hundred cells) the medium was changed from the above explant medium to modified explant medium containing wash medium containing FBS, 1% pen/strep and antifungal agent. As discussed above, this generally occurred by about day 14. The medium was then changed every other day with 10 ml of medium. Time to reach confluency depended on the number of attached pulp explants in the T-75 flask, but generally occurred about six weeks after tooth extraction.

This is a general protocol for preparation of pulp cells suitable for culture and seeding a matrix to generate constructs for use in restoration of a damaged tooth.

Example 5: Cell Seeding and Response to Three Dimensional Constructs

To identify a matrix that can support growth of pulp cells, pulp cells are seeded into a test matrix, cultured, and various properties of the cells are assayed. In one example, cells that have grown out of pulp tissue implants are harvested and are seeded in alginate: the cells are added to a syringe alone with the alginate solution (see fabrication section, supra). The cells become embedded in the alginate after the cell/alginate solution drops into a CaCl₂ solution.

To embed cells in collagen, the cell solution is added to the collagen solution after neutralization but before polymerization. The resulting cell/collagen mixture is then incubated at 37⁰C for 2 hours. Cellular attachment and growth morphology are examined using histological staining and scanning electron microscopy (SEM). At selected time points, the samples are washed three times with PBS to remove unadhered cells.

To examine morphology of the cells in a test matrix; histochemical staining is performed. For example, a cell/matrix sampleis fixed in 4% paraformaldehyde, dehydrated in ethanol, and embedded in paraffin using methods known in the art. The sample is then sectioned and stained with hematoxyline and eosin using methods known in the art.

For SEM analysis, a sample is first dehydrated using an ethanol drying series, and dried in Freon overnight in a chemical hood. Prior to imaging, the sample is coated with carbon to eliminate charging effects.

Cell proliferation is determined using the Picogreen™ dsDNA Quantitation assay (Molecular Probes, Carlsbad, CA) where fluorescence intensity is correlated to DNA concentration.

To ascertain cell phenotype and the suitability of a test matrix for promoting and maintaining differentiation of pulp cells, marker proteins or RNAs for marker proteins and mineralization within the matrix are assayed. For example, alkaline phosphatase (ALP) synthesis, osteocalcin production, and the formation of a mineralized matrix by these cells will be determined.

ALP expression is quantified using a colorimetric assay. The samples are incubated at 37°C for 30 minutes in 0.1 M Na₂CO₃ buffer containing 2 mM MgCl₂ with disodium p- nitrophenyl phosphate (pNP-PO4) as the substrate. Standard solutions are prepared by serial dilutions of 0.5 mM p-nitrophenol (pNP) in Na₂CO₃ buffer. Enzymatic activity is then expressed as total nmoles of pNP produced per min per total cell number. Absorbance is measured at 415 nm by a Spectrofluor reader (Tecan, Research Triangle Park, NC). Induction of ALP synthesis indicates that the test matrix can support differentiation and maintenance of pulp cells.

The synthesis of osteocalcin can be determined using an immunoassay (e.g., NovoCalcin™, Metra Biosystems, Inc., Mountain View, CA).

The formation of mineralized nodules is examined by SEM/EDXA, and the specific Ca/P ratio is calculated based on a hydroxyapatite standard. Mineralization can be further confirmed using Alizarin Red S (ALZ) staining specific for calcium. For ALZ staining, a sample is washed in double distilledH₂O, and incubated in 40 mM Alizarin red solution for 10 minutes. After additional washes, the matrix scaffolds are incubated in 10% cetyl pyridinium chloride for 15 minutes to solublize reacted ALZ. La this assay, serial dilutions of 1 M CaCl₂ are used as standards. ALZ concentration per cell is calculated as molar equivalent CaCl₂ divided by the average cell number. Absorbance is measured at 570 nm using a Tecan Spectrofluor system

(Tecan, Research Triangle Park, NC). The expression of osteocalcin and dentin sialophosphoprotein (DSPP) is detected by reverse transcription followed by polymerase chain reaction (RT-PCR). For this purpose, cells are released from alginate by methods known in the art using sodium citrate, and from collagen gels using collagenase digestion. Cells can be released from chitosan using chitinase digestion. Total RNA is isolated using Rneasy® kit (Qiagen, Valencia, CA). First strand cDNA is synthesized using Superscript™ (Invitrogen, Carlsbad, CA). PCR is performed using the following primer sets: osteocalcin, sense 5'-CATGAGAGCCCTCACA-3' (SEQ ID NO: 1) and antisense 5'-AGAGCGACACCCTAGAC-S ' (SEQ ID NO:2); DSPP, sense 5'- GGCAGTGACTCAAAAGGAGC-3' (SEQ ID NO:3) and antisense 5'- TCATATTTGGCAGGTTTTTCT-S ' (SEQ ID NO:4). PCR is performed for 35 cycles at an annealing temperature of 56°C. PCR products are analyzed using 1.5% agarose gel electrophoresis and visualized by staining with ethidium bromide.

Expression of osteocalcin and DSSP indicate that the test matrix can promote expression proteins expressed in differentiated cells.

Example 6: Mechanical Properties of Pulp Tissue

The mechanical properties of pulp tissue are determined utilizing unconfined compression tests 66. Coronal pulp tissue is shaped into approximately 3 mm side cubes while frozen. The mechanical properties are measured along the long axis of the tooth as well as the two perpendicular axes. Spatial variations in mechanical properties are also investigated. When possible, multiple samples will be obtained from each tooth. The tooth number as well as the location within the pulp chamber is recorded. The equipment is the same as that used for the gels except that unconfined compression is applied. Due to expected tissue inhomogenity with anatomical location and possible anisotropy, a relatively large number of samples are tested (e.g., at least about 20) per direction and/or anatomical location. These methods are suitable for all types of mammalian pulp cells, e.g., of porcine or human origin.

The mechanical properties of the pulp tissue provide a standard for desirable mechanical properties of a matrix or matrix-containing cells that are suitable for use in constructs for transplantation to a damaged tooth, i.e., to promote repair of a damaged tooth.

Cell Culture

The protocol for obtaining pulp cells from explant outgrowth has been established. Initial and continued explant attachment to the culture flask surface is necessary for outgrowth to occur. Explants that floated in culture media did not produce cells. Explant pieces that became dislodged were removed from the flask and reattached to the bottom of another flask. Outgrowth began as early as 3 days and as late as 12 days. Colonies visible by eye grew around the explants. With time, colonies also formed at non-explant sites. The cell morphology varied with culture duration from round, to stellar, to elongated. Elongated cells eventually formed an oriented structure. After significant outgrowth occurred, i.e., a few hundred cells, the time for confluency in a T-75 flask varied from three to six weeks. Time from tooth extraction to obtaining a flask of confluent cells ranged from one to two months. Trypsinizing the explant outgrowth cells yielded

IxIO⁷ cells per flask (SD=9 x 10⁶).

Outgrowth of porcine pulp tissue was initially investigated. This established parameters for protocols. After the protocol was established with porcine samples, human teeth were utilized. Porcine cells grew faster and cell morphology was slightly different than human cells. Cultured cells can be harvested and frozen using methods known in the art, generally after the first or second passage in culture.

Example ,7: Gel Fabrication and Cell Embedding

Human pulp cells were embedded in alginate gel. Cells (2.8 x 10⁶ cells/ml) were mixed with either a 1 % or 3 % wt/vol% alginate solution (Sigma, St. Louis, MO). The cell alginate solution was dropped into a stirred 100 mM CaCl₂ solution through a 21 gauge needle. Beads were removed from solution after 15 minutes and washed in PBS. The cell embedded alginate was placed in the culture media solution and stored at 37⁰C incubator with 5% CO₂. Medium was changed every two days. Human pulp cells were embedded in collagen I gel using 1.5 ml of cell suspension (3.1 x

10⁶ cells/ml) in DMEM mixed on ice with 5.0 ml of 3.1 mg/ml collagen solution (Vitrogen, Cohesion Technologies, Palo Alto, CA), 0.5 ml HEPES (25 mM) and 0.5 ml DMEM to make a final concentration of 2.0 mg/ml collagen/cell solution. Collagen/cell solution was poured into a ' square mold inside a petri dish. The dish was incubated for 2 hours and 37⁰C. After polymerization, culture medium was added. Medium was changed every two days.

Example 8: Pulp Fibroblast Culture in Collagen Gels

To demonstrate the culture of human pulp cells, non-carious premolar and third molar teeth were obtained. Pulp was extracted from the teeth and the tissue explants were cultured in DMEM supplemented with 10% FBS, non-essential amino acids (NEAA), and antibiotics

(pen/strep). After culturing as described herein, cells that had grown out of the explants were harvested, transferred to a fresh flask, and cultured.

Cultured cells were then seeded into DMEM-HEPES -MEM-Type 1 collagen gel.

Samples were prepared containing cells at 2x10⁵ cells/ml, a final collagen concentration of 2 mg/ml, and a pH of 9.0. To prepare the seeded gel (a gel matrix containing cells), the collagen and cell mixture was placed on ice and incubated for 2 hours at 37°C at 5% CO₂ to allow polymerization to occur. After the polymerization step, Culture medium (0.5 ml) was added to each well overnight. An additional 1.0 ml of medium was added then added and medium was exchanged every other day for 28 days.

Experimental Groups

Various matrix conditions were tested to identify those that are suitable for culturing pulp cells and for transplantation. One matrix condition was an unconfϊned gel matrix. For this preparation, culture wells were pre-coated with 2% bovine serum albumin (BSA) and incubated for 1 hour at 37⁰C to create a surface that prevents gel attachment. Gel formulations were then placed in the prepared wells and gelation carried out as described herein. A second matrix condition that was tested was a confined gel matrix. In this case, Thermoplast cover slips (Fisher) were placed in the culture wells in which the matrix was prepared. A 22 gauge needle was used to scratch the remaining well surfaces to create multiple sites for gel attachment. In a third matrix condition, the matrix was prepared as a partially confined gel. In this case, a gel matrix was prepared under confined conditions (described supra) and a slit was made through the center of the confined gel to create a free edge.

Test matrices prepared under the three different conditions were tested under various conditions as follows: Group A= unconfϊned collagen gel without cells, Group B= unconfϊned collagen gel with cells, Group C = partially confined collagen gel without cells, Group D = partially confined collagen gel with cells, Group E = confined collagen gel without cells, and

Group F = confined collagen gel with cells. The test matrices were then incubated in medium at 37⁰C, 5% CO₂.

For all test matrices, minimal gel contraction was observed during the first two weeks of culture. After 14 days, gels in all groups contracted and began to detach from the well surfaces. Confined gels contracted the least (18.97%), with 23.37% for the partially confined and

53.98% for the unconfined gels. Unconfϊned gels exhibited the highest rate of contraction at 3.86% versus 1.36% per day for the unconfϊned group.

Cell morphology was also examined in those matrices containing cells. In general, cells exhibited a spherical cellular morphology upon embedding in the collagen gel. By day 2, a small percentage of pulp cells began to elongate and develop orientation patterns in the loaded gels.

Pulp cells grown in the unconfined gel were found to be randomly located in the gel. By day 28, the density and number of cells increased dramatically in comparison to that of day 0, suggesting that collagen matrix I is an appropriate matrix for pulp cellular proliferation and differentiation. Cells became elongated and oriented along the free edge for the partially confined gels. Cell proliferation in confined and partially confined gels entered the plateau phase of cell growth by day 21, while that of the unconfined gels continued to increase, entering the exponential phase. These findings indicate that pulp cells in unconfined gels are actively proliferating, while those in the confined or partially confined gels may be induced to differentiate instead of proliferating.

Confined and partially confined gels visibly exhibited an increase in cells and orientation compared to the unconfined gels, indicating that conditions for pulp fibroblast growth and differentiation are favored under some degree of restriction and loading of the collagen gel.

Human pulp cells proliferated in the collagen type I matrix over time, and responded to the local mechanical environment of the gel.

These findings demonstrate that collagen gel is useful as a matrix for pulp tissue engineering. They also illustrate the use of a method for identifying a composition that is useful for culturing pulp cells, e.g. , for transplantation.

Example 9: Pulp Cell Cultures in Chitosan Hydrogel

As described above, chitosan hydrogel is a material that can be used in compositions of the present invention. Experiments were conducted to demonstrate the suitability of chitosan for this application. Specifically, pulp cells prepared as described herein were embedded in a chitosan hydrogel film by mixing 1.36mL of cell suspension (2.0 x 10⁵ cells/ml) with 8.0 ml of 2.5% chitosan solution (89.4% deacetylation, Spectrum Chemicals, Gardena, CA), and cross- linked with 40 μl of glutaraldehyde (Sigma, St. Louis, MO). Subsequently, ImI of cells plus chitosan solution was pipetted into the cell culture well (12-well plate) to allow gelation. Cell viability and morphology within the chitosan hydrogel were examined over time. As shown in

Figures 8A and 8B, pulp cells remained viable in the chitosan hydrogel. Cell number also increased over 6 days as indicated in Figures 7A and 7B. Pulp cells were evenly distributed through out the hydrogel. Cell culture can also result in the contraction of chitosan hydrogel, with gel thickness decreasing over 50% by day 3 and stabilizing afterwards. These data demonstrate that chitosan hydrogel (chitosan) is useful for culturing pulp cells, e.g., culturing pulp cells to be used in, or as, constructs for pulp regeneration and tooth repair.

Example 10: In Vitro Co-culture of Pulp Cells

A co-culture model for culturing pulp cells was developed and tested. Pulp cells embedded in chitosan hydrogel beads (cell plus chitosan) as described herein were co-cultured with a preformed monolayer of pulp cells (0.1x10⁶ cells/cm²) in a 24-well plate. The cell plus chitosan beads were formed by mixing 200 ml of cell suspension with 2.0 ml of 2.5% chitosan solution and dispensing the solution drop-wise into 22.4% w/v sodium sulfate (LabChem Inc.) solution using a 2614 gauge needle. The final cell concentration in the chitosan hydrogel was 2.OxIO⁴ cells/ml. The cell plus chitosan beads were then cultured on top of a monolayer of pulp cells over time. This co-culture model is designed to simulate the intended clinical application, where after the removal of infected pulp, a cell plus chitosan hydrogel is placed directly in the pulp chamber in direct contact with the underlying pulp tissue. The model is based on developing a context in which the interaction between the cells from the underlying pulp and the cells embedded in the hydrogel can modulate the overall cell response within the hydrogel. Such methods can be used to test the ability of different cell types, e.g., stem cells, and/or cells prepared under various conditions, for their ability to demonstrate features such as expression of differentiated proteins, cell proliferation, and cell morphology, that are suitable for use in a composition of the invention. Cell growth for both the pulp monolayer and the pulp cells embedded in chitosan beads were compared as a function of co-culture (Fig. 4 and Fig. 5, respectively). In both cases, co- culture had little effect on cell growth over the short time period examined. Cell number continued to increase in monolayer culture, and cell number remained relatively constant in the chitosan hydrogel beads, suggesting differences between two-dimensional and three-dimensional cultures.

Co-culture increased the ALP activity of monolayer pulp cells (Fig. 6) but had no significant effect on pulp cells grown in three-dimensional chitosan beads (Fig. 7). ALP activity per cell was significantly higher in three-dimensional cultures compared to the monolayer, further confirming the importance of three-dimensional culture in deterrnining cell response. These data also demonstrate that co-culture is a useful method for culturing pulp cells in two-dimensional cultures or in three-dimensional cultures.

Example 11 : Culture of Pulp Cells on Nanofiber Mesh

To demonstrate the ability of a nanofiber mesh (scaffold) to support growth of pulp cells, an electrospun mesh was prepared and used as a matrix for pulp cell culture. Briefly, an electrospun mesh was prepared using PLGA 85: 15, IV=0.66-0.80 dL/g (Alkermes, Cambridge, MA); N,N-dimethylformarnide (DMF) (surface tension (σ=mN/M; Fisher Scientific), and ethanol (σ=22.1 mN/m). Electrospinning parameters were 11 kV, needle tip/collecting plate distance of 90 mm, and a flow rate (q_v) of 1 ml/hour (syringe pump controlled). Meshes were cut into squares 1.5 cm x 1.5 cm and sterilized using uv irradiation. Human pulp fibroblasts were harvested from culture and seeded directly onto the meshes (scaffolds) and were cultured in DMEM containing 10% FBS, 1% pen/strep, and 1% NEAA. The initial seeding densities were determined using fluorometric DNA detection methods (PicoGreen®). Cultures were examined at 1 day, 7 days, and 14 days after the initiation of cultures. Cultures were examined using SEM, assayed for ALP activity (fluorometric assay); DNA quantified using PicoGreen, and statistical analyses were performed using ANOVA and Tukey-Kramer. It was found that smooth (non-beaded) fibers were formed when a solution of 60% DMF and 10% ethanol was used to generate the fibers. Cells proliferated on the scaffolds (Fig. 8) and cell ALP activity increased over time in culture (Fig. 9). In addition, by day 14, cells had elaborated abundant cell matrix on the mesh (Fig. 10). Both cell proliferation and ALP activity were increased in those cultures grown on aligned mesh compared to those grown on unaligned mesh.

These data demonstrate that electrospun mesh can be used to culture pulp cells, and that cells cultured on such a matrix can proliferate and express proteins indicative of cell differentiation. These data also demonstrate a method of testing matrices for their ability to support pulp cell growth and differentiation.

Example 11 : Mechanical Properties

Appropriate methods for determining pulp mechanical properties are useful for, e.g., identifying desirable parameters for constructs useful for transplantation. An unconfϊned compression testing apparatus was utilized (Ranly et al, J. Dent. 28:153-161, 2000). One porcine pulp tissue sample was cut (4 mm x 4 mm x 2mm) with the 2 mm axis in the buccal-Ungual direction. A tare load of 2 g was applied. With a ramp speed of 1 μm/s, deformations of 10 % and 20% were applied. For each deformation, stress-relaxation curves (force versus time) were recorded utilizing a 50 g load cell). The equilibrium modulus was determined for each applied deformation. The measured modulus was 1.3 fcPa. The significance of this experiment is that the mechanical testing equipment used is appropriate, but a 10 g load cell may increase experimental sensitivity. With a 10 g load cell a lower smaller tare loads and specimen sizes can be used.

Other Embodiments It is to be understood that while the invention has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims. Reference List l.ADA. Survey of Dental Practice. 5S99. 1999. American Dental Association. Survey of Dental Services. (GENERIC) Ref Type: Report 2. Kling, M., Cvek, M., and Mejare, L: Rate and predictability of pulp revascularization in therapeutically reimplanted permanent incisors. Endod.Dent.Traumatol. 2:83, 1986.

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Claims

WHAT IS CLAIMED IS:

1. A composition comprising a physiologically acceptable matrix seeded with pulp cells.

2. The composition of claim 1 , wherein the matrix comprises at least one agent that is an antibiotic, antifungal agent, or growth factor.

3. The composition of claim 1 , wherein the matrix comprises at least two antibiotics.

4. The composition of claim 2 or claim 3, wherein the composition comprises an antibiotic selected from the group consisting of ciprofloxacin, Minicyclin, and metronidazole.

5. The composition of claim 2, wherein the agent is time released.

6. The composition of claim 1 , wherein the physiologically acceptable matrix is capable of being injected into the pulp chamber of a tooth.

7. The composition of any one of claims 1 to 6, wherein the matrix comprises a hydrogel, a mesh, a microsphere, and a combination of any of the foregoing.

8. The composition of claim 7, wherein the matrix is a hydrogel selected from the group consisting of collagen, chitosan, alginate, MATRIGEL™, gelatin, JELL-O^®, fibrin, polyethylene glycol, and a combination of any of the foregoing.

9. The composition of claim 7, wherein the matrix is a mesh selected from the group consisting of polylactide-coglycolide (PLGA) mesh, polylactide (PLA) mesh, or polyglycolide (PGA) mesh, a cross-linked fiber mesh, a nanofiber mesh, a mesh fabric, biodegradable polymer mesh, , and a combination of any of the foregoing..

10. The composition of claim 7, wherein the matrix is a microsphere selected from the group consisting of a biodegradable polymer microsphere, a hydrogel microsphere, and a combination of any of the foregoing..

11. The composition of claim 1 , wherein the matrix comprises a nanofiber, an artificial three- dimensional scaffold material, or a synthetic three-dimensional scaffold material.

12. The composition of claim 1 , wherein the matrix comprises a polycaprolactone polymer, a polyglactan polymer, a polyanhydride polymer, or a combination of any of the foregoing.

13. The composition of claim 1 , wherein the matrix comprises type I collagen and type HI collagen.

14. The composition of claim 13, wherein the ratio of type I collagen to type III collagen is

30%:70%, 55%:45%, 45%:55%, or 70%:30%.

15. The composition of claim 1 , wherein the matrix comprises type I collagen and the collagen concentration is about 0.3% to 3.0%, about 0.3% to 0.5%, or about 0.5% to about 3.0%.

16. The composition of claim 15, wherein the gelation pH is about 6.0, 7.5, or 9.0.

17. The composition of claim 1, wherein the matrix comprises alginate and the alginate concentration is about 1.0% to 5.0%, 1.0% to 3.0%, or 3.0% to 5.0%.

18. The composition of claim 17, wherein the composition comprises CaCl₂.

19. The composition of claim 18, wherein the CaCl₂ concentration is about 50 mM, about 100 mM, or about 200 mM.

20. The composition of claiml , wherein the matrix comprises chitosan.

21. The composition of claim 1, wherein the viscosity of the gel is less that 100,000 cP at 37°C.

22. The composition of any one of claims 1 to 21 , wherein the matrix forms a scaffold upon which the pulp cells can grow.

23. The composition of any one of claims 1 to 21 , wherein the composition further comprises one or more cell growth factors.

24. The composition of any one of claims 1 to 21 , wherein the cells comprise at least one of pulp-derived stem cells, progenitor cells, or mesenchymal stem cells.

25. The composition of any one of claims 1 to 21, wherein the pulp cells are obtained from a subj ect or from a cell culture.

26. The composition of any one of claims 1 to 21 , wherein the cells are pulp cells, bone marrow cells, or a combination thereof.

27. The composition of claim 1 , wherein the matrix degrades over time.

28. The composition of claim 1 , wherein the matrix, after placement in a pulp chamber, can degrade over time.

29. The composition of claim 1 , wherein the composition comprises chitosan.

30. The composition of claim 1 , wherein the composition further comprises platelet-rich plasma-derived growth factors.

31. The composition of claim 1 , wherein the composition comprises at least one of one or more bone morphogenic proteins (BMPs) or dentin powder.

32. The composition of claim 1 , wherein the matrix is seeded with about 1 x 10⁶ cells/ml, about 2 x 10⁶ cells/ml, or about 3 x 10⁶ cells/ml.

33. A method comprising administering to a subject a physiologically acceptable matrix into the pulp chamber of a tooth.

34. The method of claim 33, wherein the physiologically acceptable matrix is seeded with cells.

35. The method of claim 33 or claim 34, wherein the composition comprises a physiologically acceptable matrix capable of being injected into the pulp chamber of a tooth.

36. The method of claim 34, wherein the pulp chamber is substantially free of native pulp cells.

37. The method of claim 33 or claim 34, wherein the pulp chamber comprises native pulp cells.

38. The method of claim 33 or claim 34, wherein the composition is inserted apically to the native pulp cells.

39. The method of claim 33 or claim 34, wherein the composition is inserted coronally to the native pulp cells.

40. The method of claim 33 or claim 34, wherein at least two different compositions are inserted into the pulp chamber.

41. The method of claim 33 or claim 34, wherein the administering comprises injection.

42. A method for treating an individual having a pulp disorder or pulp damage within the pulp chamber of a tooth, the method comprising administering a composition comprising a physiologically acceptable matrix into the pulp chamber.

43. The method of claim 42, wherein the matrix is seeded with pulp cells.

44. The method of claim 42, wherein pulp tissue is removed from the pulp chamber.

45. The method of claim 42, wherein pulp tissue is not removed from the pulp chamber.

46. The method of claim 42, wherein the cells of the composition are derived from the individual.

47. The method of claim 42, wherein the cells of the composition are not derived from the individual.

48. The method of claim 42, wherein the cells are mesenchymal stem cells, embryonic stem cells, or umbilical cord-derived cells.

49. The method of claim 42, wherein following injection of the composition, the pulp chamber is sealed.

50. A composition comprising a scaffold of electrospun collagen, electrospun PLGA, degradable polymer, or chitosan mesh, wherein the scaffold comprises at least one antibiotic or growth factor.

51. A method for culturing mesenchymal stem cells or pulp fibroblasts, the method comprising culturing the cells on the composition of claim 50.

52. A method for culturing primary pulp cells, the method comprising seeding the pulp cells that have migrated from a pulp explant in a matrix comprising collagen or a hydrogel.

53. The method of claim 52, wherein the cells are cultured in a hydrogel and the hydrogel is alginate or chitosan.

54. A kit comprising a physiologically acceptable matrix for seeding with pulpal cells and instructions for use.

55. The kit of claim 54, wherein the kit comprises a medium suitable for maintenance of harvested pulp cells.

56. The kit of claim 54, wherein the kit comprises sealant suitable for sealing a tooth.

57. The kit of claim 54, wherein the kit comprises a chamber for culturing pulp cells on a matrix.