Patent Application: US-99773401-A

Abstract:
tooth tissues include the pulp mesenchyme that forms the dentin and an epithelium that is responsible for enamel formation . cells from these tissues were obtained from porcine third molars and were seeded onto a biodegradable scaffold composed of a polyglycolic acid — polylactic acid copolymer . cell polymer constructs were then surgically implanted into the omentum of athymic nude rats so that the constructs would have a blood supply and these tissues were allowed to develop inside the rats . infrequently , columnar epithelial cells were observed as a single layer on the outside of the dentin - like matrix similar to the actual arrangement of ameloblasts over dentin during early tooth development . developing tooth tissues derived from such cell polymer constructs could eventually be surgically implanted into the gum of an edentulous recipient where the construct would receive a blood supply and develop to maturity , providing the recipient with a biological tooth replacement .

Description:
our goal is to produce a biological tooth replacement using tissue - engineering methodology based on seeding dissociated tissues onto biodegradable polymer scaffolds , and allowing the cell / polymer constructs to develop into tooth tissues inside of a suitable host . polymer scaffolds are molded in the shape of human teeth using polyvinylsiloxane molds and seeded with dissociated tissues from unerupted porcine third molars . cell / polymer constructs are implanted into the omentum of athymic rats so that the developing tooth tissues receive an adequate blood supply . cells dissociated from the enamel organ and pulp organ and cells from tissue cultures derived from tooth tissues , are seeded onto molded tooth - shaped polymers , and implanted for development in rat hosts . analysis of the resulting tooth tissues is performed using histological staining methods such as von kossa ( calcification ), goldner &# 39 ; s ( ossification ), and van gieson &# 39 ; s ( collagen ). immunohistochemical staining is also performed using antibodies specific for tooth epithelial markers ( keratin , amelogenin ) and mesenchymal markers ( osteocalcin , bone sialoprotein and dentin sialophosphoprotein ). the results of these experiments establish the identity of ameloblasts that are responsible for enamel formation , and odontoblasts that are responsible for dentin formation , within the engineered tooth tissues . immunofluorescence using the above markers is applied to cells in culture to characterize them prior to seeding on polymer scaffolds . in situ hybridization is used to detect the presence of dspp mrna , a marker for odontoblast cells , and to help distinguish between tissues of the rat host and developing porcine tooth tissues . table 1 provides an overview of the invention . table 2 provides an overview of the polymer scaffold preparation . chemicals . polyglycolic acid ( pga ), poly - l - lactic acid ( plla ), poly - l - lactide - co - glycolide ( plga ), chloroform , dichloromethane , polyvinylsiloxane dental impression material ( reprosil ), sodium chloride , hank &# 39 ; s balanced salt solution ( hbss ), phosphate - buffered saline solution ( pbs ), dulbecco &# 39 ; s modified eagle medium ( dmem ), fetal bovine serum , glutamax , penicillin , streptomycin , sorbitol , 0 . 9 % saline solution , iodine solution ( povidine ), 70 % ethanol , collagen ( type i ), 0 . 01 m hydrochloric acid , collagenase , dispase , ketamine , xylazine ( rompun ). tissues . human incisors and molars , six - month - old porcine third molar tooth tissue . preparation of tooth molds . extracted human incisors and molars were used to cast negative impression tooth molds in polyvinylsiloxane dental impression material ( reprosil ). once the impression material hardened , the teeth were removed by cutting an opening in one side of the mold with a razor blade . this method leaves a tooth - shaped cavity inside the impression material which can be filled with polymer solution for the preparation of biodegradable tooth scaffolds . preparation of polymer tooth scaffolds . pga mesh material was broken up into 1 - 2 mm flakes and packed into the cavity of a tooth mold to fill it completely . the remainder of the cavity volume was filled with a 3 % w / w plla solution in chloroform . the pga / plla mixture was heated to approximately 400 ° f . for 5 minutes to bond the two polymers and then lyophilized for 48 h . for plga tooth scaffolds , plga crystals were first dissolved in chloroform to 5 % w / w . negative tooth molds were packed to half - capacity with sodium chloride crystals ( 75 - 150 μm ) and the remainder of the mold volume was filled with 5 % plga solution . sodium chloride crystals were added to create a thick slush in the plga solution and the mixture was lyophilized for 48 h . scaffolds were removed from the molds and placed in distilled water for 24 h to leach out the salt crystals leaving behind a porous plga sponge material in the shape of a tooth . tissue dissociation . fresh pig jaw dissected from a freshly slaughtered six - month - old pig was placed on ice for transport . the jaw was split in two and muscle and connective tissue were removed from the bone using a razor blade . a dental drill fitted with a spherical bit was used to drill holes along the lingual side of the bone surface along lines adjacent to the regions harboring the 3 rd and 2 nd unerupted molars . bone chisels were used to break the bone in between the drilled holes and then the resulting bone flap was pried and lifted away to expose the unerupted molars . a dental probe was used to carefully lift out the 3 rd molar and connective tissue was cut with surgical scissors . the molars were placed in ˜ 50 ml of hank &# 39 ; s balanced salt solution ( hbss ) and kept at 4 ° c . in 50 ml sterile conical tubes . prior to mincing the tissues , the immature tooth cusps were removed and discarded . the remaining enamel and pulp organ tissues were minced into 2 - 3 mm 3 pieces in a sterile petri plate in hbss . tissues were washed 5 times in hbss , minced into & lt ; 1 mm 3 pieces and then treated with 1 . 5 units of vibrio alginolyticus collagenase and 12 units of bacillus polymyxa dispase for 25 minutes at room temperature . gentle mechanical dissociation of tissues was achieved by pipetting the suspension up and down in a 25 ml pipette for 10 min followed by 15 min with a 10 ml pipette . tissues were washed five times in dmem ( containing 2 . 5 % fbs , 2 % sorbitol , glutamax , 50 units / ml penicillin , 50 □ g / ml streptomycin ) and then cells were counted using a hemacytometer . typical cell yields were 2 . 0 × 10 6 cells / ml . seeding of biodegradable polymer scaffolds . pga / plla and plga tooth scaffolds were coated with collagen overnight at 4 ° c . in a 1 mg / ml type i collagen solution in 0 . 01 m hcl . scaffolds were washed three times in pbs then three times in dmem + supplements ( see above ). ˜ 2 . 0 × 10 6 cells were seeded onto each tooth scaffold and cells were given at least 1 hour to attach . laparotomies were performed on athymic nude rats and seeded scaffolds were implanted into the omentum to provide a blood source for the developing tooth tissues . tissues were allowed to develop inside the host animals for 7 - 20 weeks before they were sacrificed and the engineered tissues harvested . the results of these experiments are generally summarized in table 4 . polymer tooth scaffolds . human incisors and molars were used to make negative impressions in reprosil dental impression material . pga mesh material was then broken into flakes and these flakes were used to completely fill the tooth mold . the remainder of the mold cavity volume was filled with a 3 % plla solution in chloroform . the pga / plla mixture was heated to approximately 400 ° f . for 5 minutes to bond the two polymers and then lyophilized for 48 hours . the plga tooth scaffolds were made as described in the materials and methods section . tooth tissues . immediately after slaughter , mandibles from six - month old pigs were collected at the slaughterhouse and transported on ice to the laboratory . the second and third unerupted molars from each hemimandible were dissected from the jawbone and were immersed in separate vials containing hanks balanced salt solution . all teeth were kept at 4 ° c . prior to dissociation of the tissues . the enamel and pulp organ tissues were minced , washed , minced again , and treated with collagenase / dispace in order to obtain the greatest amount of single cells in suspension . the cells were then washed several more times and were resuspended in dmem containing 2 . 5 % fbs and 2 % sorbitol . seeding of biodegradable polymer scaffolds . pga / plla and plga tooth scaffolds were coated with collagen overnight at 4 ° c . in a type i collagen solution in 0 . 01 m hcl . scaffolds were washed three times in pbs then three times in dmem plus fbs and sorbitol . cells were seeded onto each tooth scaffold and were given at least 1 h to attach . laparotomies were performed on athymic nude rats and seeded scaffolds were implanted into the omentum to provide a blood source for the developing tooth tissues . the tissues were allowed to develop inside the host animals for 7 - 20 weeks before they were sacrificed and the engineered tissues harvested . characterization of bioengineered tooth tissues . host animals were sacrificed after 7 . 5 weeks of development and the tooth tissues were dissected , preserved and fixed in formalin , and embedded in paraffin for histological sectioning . tissue sections were stained with hematoxylin and eosin and counterstained by the von kossa method to identify mineralized tissues . tissues were also stained by the method of van gieson to identify areas of ossification and were stained by the method of goldner to detect the presence of collagen . histological sections of engineered tooth tissues revealed an organization analogous to the early tooth bud . present was a layer of collagenous matrix that appeared similar to that observed in dentin or bone . the surrounding region of mesenchyme looked like what is observed in pulp tissue . infrequently , a single layer of columnar epithelium was observed on the outer face of the collagenous matrix , resembling epithelial ameloblast cells , which form dental enamel . some regions of the collagenous matrix stained positively for the presence of calcified mineral deposits suggesting that biomineralization had occurred . preliminary results demonstrate successful use of porcine odontogenic cells to generate replacement molar and incisor teeth . mineralization was observed in a four week - old tissue culture of the dissociated porcine third molar tissues , suggesting that a mixture of dissociated tooth tissue cells can spatially reorganize themselves in vitro , and generate calcified deposits . dissociated porcine tooth tissue cells were seeded onto collagen - coated pga scaffolds and implanted into the omenta of rat hosts . histological analysis of 7 . 5 - week - old implanted pga scaffolds revealed an organization similar to that of the early tooth bud . a layer of collagenous matrix similar to dentin or bone surrounded the mesenchyme tissue . this resembles what naturally occurs in pulp tissue . rarely , a single layer of columnar epithelium was observed on the outer face of the collagenous matrix . this is similar to the organization of enamel - forming epithelium just prior to the formation of dental enamel . the presence of calcified mineral deposits suggests that biomineralization had occurred . these results demonstrate that it is possible to grow mineralized tooth tissues using biodegradable polymer tooth scaffolds seeded with tooth bud cells . thus , we have demonstrated that by use of the tissue engineering techniques described here , it is possible to grow mineralizing tissues that resemble those of the developing tooth . rationale . tooth molds are used to prepare scaffolds in the shapes of individual human teeth so that the seeded tooth cells will form a tooth of a predetermined shape and size . experimental approach . extracted human incisors and molars are used to create tooth molds in polyvinylsiloxane dental impression material ( reprosil ). once the impression material has hardened , the teeth are removed by cutting an opening in one side of the mold with a razor blade . this method leaves a tooth - shaped cavity inside the impression material , which can be filled with polymer solution for the preparation of biodegradable tooth scaffolds . rationale . polymer scaffolds of optimal porosity are necessary so that the seeded cells can migrate through the scaffold to their appropriate positions to begin forming the tissue - engineered tooth . experimental approach . pga mesh material is broken into 1 mm 2 flakes and packed into the cavity of a tooth mold to fill it completely . the remainder of the cavity volume is filled with a 3 % w / w plla solution in chloroform . the pga / plla mixture is heated to approximately 400 ° f . for 5 min to bond the two polymers and then is lyophilized for 48 h . for plga tooth scaffolds , plga crystals will first be dissolved in chloroform to 5 % w / w concentration . tooth molds are packed to half - capacity with sodium chloride crystals ( 75 - 150 μm ) and the remainder of the mold volume is filled with 5 % plga solution . sodium chloride crystals are added to create a thick plga slush , and the mixture is lyophilized for 48 h . scaffolds are removed from the molds and placed in distilled water for 24 h to dissolve salt crystals , leaving behind a porous plga sponge material in the shape of a tooth . isolation and preparation of porcine tooth tissues for seeding onto biodegradable polymer scaffolds rationale . optimized procedures for dissociating tooth tissues for seeding onto the polymer scaffold are necessary so that both the epithelial and mesenchymal cells will attach , migrate to their appropriate positions , and form their respective mineralized tissues ( enamel and dentin ). experimental approach . a jaw dissected from a freshly slaughtered six - month old pig is placed on ice for transport from the farm ( athol , mass .) to the forsyth institute . the jaw is split at the midline . muscle and connective tissue are removed from the bone using a razor blade . a dental drill fitted with a spherical burr is used to drill holes along the jaw ( lingual side ) surrounding the region of the m2 and m3 unerupted molars . bone chisels are then used to break the bone in between the drilled holes , and the resulting bone flap is removed to expose the unerupted molars . a dental probe is used to carefully lift out m2 and m3 tooth sacs , and the connective tissue is removed with surgical scissors . the molars are placed in ˜ 50 ml of hank &# 39 ; s balanced salt solution ( hbss ) and kept at 4 ° c . in 50 ml sterile conical tubes . when present , immature tooth cusps are removed and discarded , and the remaining enamel and pulp organ tissues are minced into 2 - 3 mm 3 pieces in a sterile petri dish in hbss . tissues are washed 5 times in hbss , minced into & lt ; 1 mm 3 pieces and treated with 1 . 5 units of vibrio alginolyticus collagenase and 12 units of bacillus polymyxa dispase for 25 min at room temperature . gentle mechanical dissociation of tissues is achieved by gentle pipetting with a 25 ml pipette for 10 min followed by 15 min with a 10 ml pipette . the dispersed cells are washed five times in dmem ( containing 2 . 5 % fbs , 2 % sorbitol , glutamax , 50 units / ml penicillin , 50 μg / ml streptomycin ) and pelleted by gentle centrifugation at approximately 400 × g and counted using a hemacytometer . typical cell yields are approximately 5 . 0 × 10 6 cells / ml . molded pga / plla and plga tooth scaffolds are coated with collagen overnight at 4 ° c . in a 1 mg / ml type i collagen solution in 0 . 01 m hcl . scaffolds are washed three times in pbs then three times in dmem + supplements ( see above ). approximately 2 . 0 × 10 6 cells are seeded onto each tooth scaffold and allowed to attach for at least 1 hour . laparotomies are performed on athymic nude rats and the seeded scaffolds are implanted into the omentum , providing a blood source for developing tooth tissues . the implants are allowed to develop inside the host animals for 7 - 35 weeks before the engineered tissues are harvested . to our knowledge , no study has ever examined the feasibility of growing biological teeth using dissociated tooth tissues seeded on biodegradable polymer scaffolds . one group has used hydroxyapatite / tricalcium phosphate powder mixed with cultured dental pulp cells to generate a small amount dentin matrix secreted by odontoblast - like cells six weeks after subcutaneous implantation in nude mice ( gronthos et al ., 2000 ). however , using our approach we have obtained structures resembling developing and mature teeth with dentin secreted by odontoblasts , enamel secreted by ameloblasts , a well - defined pulp chamber and putative cementoblasts embedded in a cementum matrix ( see preliminary data ). thus , our approach has demonstrated that it is possible to engineer developmentally advanced tooth tissues . in vitro analysis of dissociated tooth tissues . six - month old porcine third molars ( m3 ) were dissociated into cell suspensions and grown in culture for a period of four weeks . the cell cultures exhibited extensive mineralization , as measured by von kossa staining ( data not shown ), suggesting that the dissociated tooth tissues could spatially reorganize themselves in vitro to form calcified deposits . twenty week implant . dissociated enamel and pulp cells obtained from a 6 month - old pig third molar were seeded onto a pga scaffold molded in the shape of a human incisor of approximately 1 cm by 0 . 5 cm in size . the cell / polymer construct was implanted into the omentum of a nude rat host and allowed to develop for 20 weeks . at this time , histological analysis revealed small tooth - shaped tissues within the implant , which were similar in appearance to that of a very small cusp tip ( fig1 ). we observed mineralized dentin - like tissue ( d ) and beneath the dentin , was a pre - dentin - like layer ( pd ) that appeared to be secreted by odontoblast - like cells ( o ). vascularized mesenchyme resembling that of pulp tissue ( pt ) filled the remainder of the pulp cavity ( fig1 ). the cellular organization of another 20 week implant clearly resembles that of an early bell stage tooth bud ( fig1 a - b ). the tooth tissue was ˜ 2 mm in diameter and exhibited distinct coronal and apical organization , with recognizable cusps and root tips . putative odontoblasts ( o ) lined the inner surface of an apparently collagenous dentin matrix ( d ) ( fig1 a - b ) and putative hertwig &# 39 ; s root sheath epithelia ( h ) was also present adjacent to the developing root tips ( fig1 b ). in summary , the 20 - week tooth tissues contained putative pre - dentin and mineralized dentin components , and vascularized mesenchymal cells resembling pulp tissue populated the pulp chamber . in this twenty - week implant no ameloblast - like epithelial cells were observed on the outer face of the putative dentin tissue . twenty - five week implant . a tooth tissue implant consisting of a pga polymer scaffold molded in the shape of a human incisor seeded with ˜ 2 . 0 × 10 6 porcine tooth tissue cells was dissected from a nude rat at 25 weeks post implantation . the tissue was fixed in neutral formalin , embedded in paraffin , sectioned and then stained with hematoxylin and eosin ( fig1 a ). a tooth bud with a diameter of 2 mm was discovered within the excised tissue . the interior core of the tooth bud consisted of pulp - like mesenchymal cells lined with columnar odontoblasts which were adjacent to a dentin - like layer , as has been observed in previous 20 - week tooth buds ( see fig1 and 18 a - b ). in some locations a different mineralizing layer ( e , em ) was observed ( fig1 a ) which closely resembled decalcified porcine enamel . a darkly stained region was found directly adjacent to numerous columnar cells possessing polarized nuclei which closely resembled ameloblasts ( a ). this densely stained region was thought to be enamel matrix since the staining was significantly reduced in the deeper layers just as it is for naturally forming enamel . the same tissue was stained by the method of goldner ( fig1 b ) which stains osseous tissues blue - green while the mature dental enamel stains a bright red color ( dr . ziedonis skobe , personal communication ) thus , after 25 weeks of implant development , we have obtained engineered tissues that are composed of the two major mineralizing structures of the tooth : the dentin and enamel . thirty week implant . anatomy of an inverted tooth . porcine tooth tissues were seeded onto a plga scaffold which was implanted into a nude rat and harvested 30 - weeks later . fig2 a - c show a demineralized , hemotoxylin - stained section from the implant . fig2 a shows a layer of dentin that surrounds a thick layer of enamel . a close - up reveals the unmistakable columnar rows of ameloblasts ( a ) with polarized nuclei ( fig2 b ). the cellular tissue adjacent to the ameloblasts is morphologically similar to the stratum intermedium and the remaining cellular tissue is very similar to the stellate reticulum . thus , it appears that the three major tissue morphologies of the enamel organ are also present within this inverted tissue engineered tooth . even more striking , was the appearance of putative cementoblasts ( c ) that were embedded within their own matrix ( fig2 c ). thus , although this is an inverted tissue - engineered tooth , this tooth appears to be developing all the necessary components of a healthy maturing tooth . we believe that this small tooth structure ( approximately 2 mm in length ) is inverted because not enough cells were originally seeded onto the scaffold . previous studies demonstrate that approximately , 20 - 50 million cells are required per square cm for engineered tissues to conform to the shape of the scaffold and this implant had approximately 10 fold less cells than was required . in a separate set of experiments designed to confirm the identity of the engineered tissues , we performed immunohistochemical analysis using antibodies specific for proteins present in epithelia , dentin / bone , or enamel . immunohistochemical analysis of decalcified porcine m3 control teeth with an anti - pancytokeratin antibody resulted in staining of porcine ameloblasts and stratum intermedium cells , but no staining of the odontoblasts . the same antibody reacted relatively strongly with rat epithelial cells present in sectioned rat mandible tissue . we are evaluating engineered tooth tissues by immunohistochemical staining with the anti - pancytokeratin antibody as well as antibodies against amelogenin , osteocalcin , bone sialoprotein , and dentin sialophosphoprotein ( dspp ). we are currently performing in situ hybridization analysis for the dentin - specific protein dspp to confirm the identity of the odontoblasts and dentin tissues . these marker analyses will help identify ameloblasts , odontoblasts , and cementoblasts present in the tissue - engineered tooth tissues . in conclusion , we have demonstrated successful engineering of recognizable teeth , using biodegradable polymer scaffolds seeded with porcine third molar tooth cells . the teeth form dentin from cells appearing to be odontoblasts , have a well defined pulp chamber , possess putative hertwig &# 39 ; s root sheath epithelial , possess putative cementoblasts , and have a morphologically correct enamel organ consisting of stellate reticulum , stratum intermedium , and ameloblasts , and have what appears to be fully formed dental enamel . baba t , terashima t , oida s , sasaki s . 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