Tyrofill-titanium implant constructs for the coordinated repair of mandible and tooth defects

Abstract

A composition tor use in treating a dental implant. Tire composition comprises a porous biodegradable polymer scaffold and a dicalciurn phosphate dihydrate (DCPD) coating within pores of the scaffold.

Description

TYROFILL-TITANIUM IMPLANT CONSTRUCTS FOR THE COORDINATED REPAIR OF MANDIBLE AND TOOTH DEFECTS CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to and benefit of U.S. Provisional Patent Application No. 63 / 544,361, filed October 16, 2023, which is hereby incorporated by reference herein in its entirety. GOVERNMENT SUPPORT

[0002] This invention was made with government support under grant no AFIRM II CF-04, W81-XWH-14-2-0004, the Bioengineered Alveolar Bone and Tooth Constructs, and NIH / NIDCR / NIBIB R01DE026731. The government has certain rights in the invention. TECHNICAL FIELD

[0003] The present invention relates to compositions and methods for treating a dental implant. More specifically, the compositions and methods include use of a porous biodegradable polymer scaffold (e.g., a modified TyroFill [E1001(1K) / dicalcium phosphate dihydrate (DCPD)] scaffold) and a dicalcium phosphate dihydrate (DCPD) coating within pores of the scaffold to support titanium dental implant model for simultaneous bone-dental implant repair. BACKGROUND

[0004] Craniomaxillofacial (CMF) defects remain a significant health concern where trauma, cancer, and birth defects can cause significant physical and psychological impacts. Critical-sized CMF defects cannot heal on their own due to their limited regenerative potential. Therefore, the repair of CMF defects requires highly specialized surgical interventions to restore proper form and function. Currently used CMF repair therapies require a lengthy, multi-staged surgical approach first for bone augmentation, followed by surgical dental implant placement. One of the main challenges for these therapies is to ensure the successful regeneration of sufficient bone to provide reliable support for the dental implant. Autologous bone grafting remains the gold standard for CMF defect repair therapies, based on its superior properties with respect to immune response and biocompatibility. Clinical applications for bone repair by xenograft and allograft therapies are limited by concerns regarding potential immune rejection, exposure to viral contaminants, inadequate bone regeneration, donor site morbidity, and unpredictable bone graft survival. To date, tissue engineering approaches using autologous bone-forming cells are considered to be the most promising therapies for effective boney defect repair.

[0005] Currently used methods to repair craniomaxillofacial (CMF) bone and tooth defects require a multi-staged surgical approach for bone repair followed by dental implant placement. Previously published results demonstrated significant bioengineered bone formation using human dental pulp stem cell (hDPSC) seeded tyrosine-derived polycarbonate scaffolds [E1001(1K)- bTCP].

[0006] It is desirable to develop more effective therapies for the coordinated regeneration and functional repair of CMF defects that reduce costs and patient recovery times. SUMMARY

[0007] According to aspects of the present disclosure, a composition for use in treating a dental implant is disclosed. The composition comprises a porous biodegradable polymer scaffold and a dicalcium phosphate dihydrate (DCPD) coating within pores of the scaffold.

[0008] According to other aspects of the present disclosure, a method of forming a composition for use in treating a dental implant is disclosed. The method comprises coating the pores of an E1001(1K) scaffold with dicalcium phosphate dihydrate (DCPD) to form a E1001(1K) / dicalcium phosphate dihydrate (DCPD) scaffold.

[0009] According to other aspects of the present disclosure, a method of a treating dental implant is disclosed. The method comprises immersing at least a portion of the implant into a scaffold comprising E100(1K) / dicalcium phosphate dihydrate (DCPD).

[0010] These and other capabilities of the inventions, along with the inventions themselves, will be more fully understood after a review of the following figures, detailed description, and claims. BRIEF DESCRIPTION OF THE DRAWINGS

[0011] While the invention is susceptible to various modifications and alternative forms, specific embodiments have been shown by way of example in the drawings and will be described in detail herein. It should be understood, however, that the invention is not intended to be limited to the particular forms disclosed. Rather, the invention is to cover all modifications, equivalents, and alternatives falling within the spirit and scope of the invention as defined by the appended claims.

[0012] FIG.1 shows a detailed schematic of an in vivo TyroFill-Ti implant study design, where the black circle indicates the location of a rabbit mandible defect site.

[0013] FIG.2A shows hDPSCs morphologies prior to cell seeding.

[0014] FIG.2B shows HUVECs morphologies prior to cell seeding.

[0015] FIG. 2C shows TyroFill-Ti constructs after 1-week in vitro culture in OM, with cells (upper panel) or without cells (lower panel).

[0016] FIG. 2D shows a histological analysis demonstrating abundant cell distribution throughout the TyroFill construct before implantation.

[0017] FIG. 2E shows double IF staining revealing both hDPSCs and HUVECs in Tyrofill implants prior to implantation.

[0018] FIG. 2F shows a statistical analysis demonstrating hDPSCs and HUVEC retained an ~1:1 ratio prior to implantation.

[0019] FIG. 3A shows representative 3-D μCT images of harvested 1-month (upper panel) and 3-month (lower panel) implants.

[0020] FIG. 3B shows quantification of new bone volume / tissue volume (BV / TV) and trabecular thickness within the constructs at the implant site.

[0021] FIG.4A shows H&E stained 1-month hDPSCs / HUVEC seeded constructs.

[0022] FIG.4B shows acellular TyroFill constructs.

[0023] FIG.4C shows 3-month hDPSCs / HUVEC seeded constructs.

[0024] FIG.4D shows acellular TyroFill constructs.

[0025] FIG. 5 shows immunofluorescent analyses of bone differentiation marker expression in bioengineered bone TyroFill constructs.

[0026] FIG. 6A shows an SEM analysis of Ti dental implants removed from harvested TyroFill constructs.

[0027] FIG. 6B shows an EDAX analysis of Ti dental implants removed from harvested TyroFill constructs after 1 month implantation.

[0028] FIG.6C shows another EDAX analysis of Ti dental implants removed from harvested TyroFill constructs after 3 months implantation.

[0029] While the invention is susceptible to various modifications and alternative forms, specific forms thereof have been shown by way of example in the drawings and will herein be described in detail. It should be understood, however, that it is not intended to limit the invention to the particular forms disclosed, but, on the contrary, the intention is to cover all modifications, equivalents, and alternatives falling within the spirit and scope of the invention. DETAILED DESCRIPTION

[0030] While the inventions described herein are susceptible of embodiment in many different forms, there is shown in the drawings and will herein be described in detail preferred embodiments of the inventions with the understanding that the present disclosure is to beconsidered as an exemplification of the principles of the inventions and is not intended to limit the broad aspects of the inventions to the embodiments illustrated.

[0031] As described in detail herein, a new model was tested for coordinated, simultaneous jawbone-dental implant placement and regeneration, using the Kohn laboratory fabricated E1001(1k)-DCPD scaffolds (now referred to as TyroFill scaffolds) supporting a titanium dental implant, in a critical sized rabbit mandible defect repair model.

[0032] A unique consideration for repairing the CMF complex is the fact that craniofacial bones have a distinct embryological origin as compared to the axial and appendicular skeleton. Craniofacial bones, including the jaw bones, arise from neurectoderm derived neural crest cells, while other bones are of mesodermal origin. CMF bones, including the tooth supporting alveolar bone that resists resorption in response to the strong forces of mastication, differ from axial and appendicular bones with respect to their response to mechanical and homeostatic stimuli. Moreover, many CMF bones undergo intramembranous as opposed to endochondral ossification. Craniofacial bone grafts show improved volumetric maintenance and survival when used in CMF repair, although bone marrow derived mesenchymal stem cells (MSCs) derived from mesoderm are most commonly used for craniofacial bone regeneration. This is due to the relative scarcity of CMF bones and the likelihood of disfigurement by harvesting procedures. Human dental pulp stem cells (hDPSCs), which can be harvested from extracted deciduous, wisdom, and other teeth, have the distinct capability to not only form tooth related tissues, but also mineralized tissues exhibiting characteristics of alveolar bone. Advantages for using hDPSCs for CMF tissue repair include the fact that they share the same embryonic tissue origin - they are both derived from the neural crest – and hDPSCs can easily be harvested from teeth that would otherwise be discarded. In addition, hDPSCs have the demonstrated capacity to regenerate a variety of craniofacial tissues, including composite jawbone and dental tissues.

[0033] Another important consideration for tissue engineered regenerative therapies is the type of scaffold to select for large defect repair. Synthetic polymer / ceramic scaffolds are some of the most commonly used scaffolds for bone regeneration due to the fact that they are biomimetic and osteoinductive. Tyrosine-derived polycarbonate (TyrPCs) scaffolds, recently developed by the Kohn laboratory, have been extensively characterized for applications in bone regeneration. To date, TyrPC family derived porous scaffolds fabricated from 90 mol% DTE, 10 mol% DT, and 1 mol% PEG (MW=1 kDa), abbreviated as E1001(1k), have been shown to support robust bone regeneration in calvarial and long bone defect repair models, particularly when the E1001(1k) scaffolds included calcium phosphate. Moreover, more soluble forms of calcium phosphates for bone scaffold fabrication have been widely investigated for bone engineering strategies. Basedon these promising properties, the utility of hDPSC-seeded E1001(1k)-bTCP scaffolds for alveolar bone regeneration in a small animal rat ramus defect repair model was investigated. Based on promising results in the rat, tyrosine-derived polycarbonate E1001(1K)-bTCP scaffolds seeded with hDPSCs and Human Umbilical Vein Endothelial Cells (HUVECs) were then demonstrated to support the formation of abundant alveolar jawbone regeneration in a critical- sized rabbit mandible defect repair model. In the rabbit study, active bone remodeling by both osteoblasts and osteoclasts present were observed on newly formed bone surfaces in all implants and, in particular, the implants seeded with human DPSC / HUVECs. Together, these studies demonstrated the utility of hDPSCs-seeded E1001(1K)-bTCP scaffolds for superior bioengineered alveolar bone regeneration. These studies also show that the rabbit mandible and tooth defect repair model serves as a robust, mid-sized animal model for human craniomaxillofacial regenerative therapies. Limitations to the rabbit model include the fact that only one 10 mm full thickness defect can made in each rabbit, limiting sample size. Rabbit studies allow for in vivo validation prior to conducting clinically relevant studies in a large animal minipig craniofacial defect repair model.

[0034] Next, determining whether further modification of E1001(1k)-bTCP scaffolds couldimprove their bone regeneration properties enough to support a dental implant was desired. A ss u c h , bTCP was replaced with a coating of dicalcium phosphate dihydrate (DCPD,CaHPO4.2H2O), also known as brushite, which is known to facilitate improved bone metabolism. The Kohn laboratory fabricated E1001(1k)-DCPD scaffolds (now referred to as TyroFill scaffolds) and demonstrated that TyroFill supports bone formation when transplanted into non-load bearing rabbit calvarial defects. The in vitro behavior of hDPSCs-seeded TyroFill scaffolds was then characterized, demonstrating comparable biocompatibility and osteoconductivity of hDPSCs as compared to hDPSCs-seeded E1001(1K)-bTCP scaffolds.

[0035] As described in detail herein, a modified TyroFill [E1001(1K) / dicalcium phosphate dihydrate (DCPD)] scaffold supported titanium dental implant model was used for simultaneous bone-dental implant repair. TyroFill scaffolds containing an embedded titanium implant, with (n=3 each time point) or without (n=2 each time point) seeded hDPCs and Human Umbilical Vein Endothelial Cells (HUVECs), were cultured in vitro. Each implant was then implanted into a 10 mm full-thickness critical-sized defect prepared on a rabbit mandibulee. After 1 and 3 months, replicate constructs were harvested and analyzed using Micro-CT histological and IHC analyses. The results showed significant new bone formation surrounding the titanium implants in cell-seeded TyroFill constructs. This study indicates the potential utility of hDPSC / HUVEC seeded TyroFill scaffolds for coordinated CMF bone-dental implant repair.

[0036] Materials and methods

[0037] Preparation of TyroFill (E1001(1k)-DCPD) scaffolds

[0038] The fabrication of E1001(1k)-DCPD (here referred to as TyroFill) scaffolds consistsof the following steps - porous E1001(1k) scaffolds were prepared by porogen leaching followedby the formation of a DCPD coating within the pores of the scaffold.

[0039] Step 1: Briefly, 2 g of E1001(1k) polymer was dissolved in 1.4 mL of deionized (DI) water and 8.6 mL of 1,4-dioxane overnight. 18 g of NaCl (particle size 212 – 425 m) were placed into a Teflon mold. The polymer solution was then slowly poured over the NaCl and allowed to diffuse throughout the salt bed for 1 hour. The Teflon mold was covered during that time. After 1 hour, the Teflon mold was frozen rapidly in liquid nitrogen and then freeze-dried for 48 h. Disk-shaped scaffolds (10 mm diameter, 6 mm height) were punched out from the Teflon mold. These disks were kept in DI water overnight to leach out the salt crystals. The leached scaffolds were dried in a leophylizer for 24 hours.

[0040] Step 2: In the second step, TyroFill scaffolds were prepared by immersing E1001(1k)scaffoldsin 1 M CaCl2solution. To ensure that the solution fills the entire pore volume, theimmersedscaffolds were first exposed to a vacuum of up to 30 inHg for 1 min followed by rapidrelease to atmospheric pressure. This was repeated 5 times. Next, the scaffolds wereimmersed in 1 MK2HPO4solution and the vacuum treatment was repeated 5 times as before.The scaffolds were alternated in CaCl2and K2HPO4solutions for 3 cycles. This resulted in theformation of a coatingof DCPD throughout the pore volume of the scaffold. The resultingTyroFill (E1001(1k)-DCPD) scaffolds were dried, placed in ETO sterilization pouches and sterilized using an ethylene oxide (EtO) sterilizer (AN74i, Anderson Products, Haw River, NC). After sterilization, the sealed scaffolds were stored at -20 °C until use.

[0041] Cell seeding

[0042] Before cell seeding, TyroFill scaffolds were pretreated in mesenchymal cell Osteogenic Media (OM) [DMEM / F12, 10% FBS, 1% GlutaMAX, 100 nM dexamethasone, 10 mM beta- glycerolphosphate, 50 μg / ml ascorbic acid, and 1% penicillin / streptomycin / amphotericin (PSA)] for one week, to further develop a calcium coating.

[0043] hDPCs were harvested and characterized as previously described. Briefly, teeth were extracted by trained clinicians at the Tufts University School of Dental Medicine (TUSDM) using Tufts University IRB approved protocols. The dental pulp was then harvested from the extracted teeth, minced into small pieces, and digested using 0.4 mg / mL collagenase type I (Sigma- Aldrich, St. Louis, MO, USA) and 0.2 mg / mL dispase (Boehringer Mannheim, Indianapolis, IN, USA) to generate single cell suspensions. hDPSCs were in vitro cultured andexpanded in 5% CO2 at 37°C in dental mesenchymal cell medium with DMEM / F12, 10% FBS, 1% GlutaMAX, 25 μg / ml ascorbic acid, and 1% PSA, and then cryopreserved until use. The multipotent (osteogenic, chondrogenic, adipogenic and neurogenic) differentiation potential of each hDPSC cell line was confirmed prior to use. HUVECs were expanded in vascular basal media (PCS100030, ATCC) with VEGF growth kit (PCS100041, ATCC) in 5% CO2 at 37°C, and cryopreserved at passage three.

[0044] Both types of cryopreserved cells were thawed and expanded in vitro immediately prior to implant fabrication. Equal numbers (1:1) of hDPSCs and Human Umbilical Vein Endothelial Cells (HUVECs, ATCC, PSC100010, Manassas, VA) were seeded dynamically ontoTyroFill scaffolds for a final density of 0.25 x 105cells / mm3. HUVECS were used to facilitatevascularization of the implanted construct. Cell-seeded and unseeded acellular scaffolds were in vitro cultured in 1:1 DPSC:HUVEC medium with osteogenic supplements listed above for one week prior to in vivo implantation to ensure sufficient time for cell attachment, proliferation and to initiate differentiation prior to implantation. Titanium (Ti) implants (SLActive® 8, Straumann, Andover, MA) were screwed into each TyroFill scaffold and cultured for an additional three days prior to implantation. Since the Ti implants used in this study had an expired sterilization date, all implants were autoclaved prior to their use.

[0045] Rabbit mandible defect repair model

[0046] All animal experiments were conducted under the guidance and approval of the Institutional Animal Care and Use Committee (IACUC) of Tufts University. The rabbit mandible defect repair model used in this study was performed on New Zealand White Rabbits (>3.5 kg). For each time point (1- and 3-months implantation), experimental samples consisted of Tyrofill scaffolds containing implants that were cell-seeded (n=3) or acellular (n=2), one implant per rabbit, 5 rabbits per time point (Figure 1A). To achieve a 95% confidence level, and 10% standard deviation, 5 animals per time point (n=2) were used. Briefly, fully anesthetized rabbits were placed in a dorsal position, and a midline incision was made under the chin, followed by the dissection of the fascia and muscle to expose the left side mandibular bone. A full thickness mandibular bone defect was made through the roots of the second molar using a 10 mm trephine bur under copious sterile saline irrigation. Buccal cortex bone, exposed tooth roots, and lingual cortex bone were sequentially removed with a periosteal elevator to create a full thickness defect, and the defect site was thoroughly irrigated with sterile saline to remove any remaining bone and tooth fragments. Next, a cell-seeded or acellular TyroFill with dental implant was placed into the defect, and 4–0 Vicryl was used to close the overlying muscle and skin layers. Heart rate, oxygen saturation, carbon dioxide, respiratory rate, and body temperature weremonitored carefully throughout the procedure. Soft, Critical Care diet was provided to experimental rabbits for 2 weeks post-operation. After 1- or 3-months implantation, implanted and contralateral unoperated control jaws were harvested using formalin perfusion to ensure sufficient fixation of implant tissues. The harvested mandibles were then re-fixed in 4% formalin for 3-5 days with rocking at room temperature, hemi-sected using a band saw, analyzed via Micro-CT, demineralized, and processed for histological and immunohistochemical analyses.

[0047] Evaluation of bioengineered mandibular bone implants

[0048] Harvested hemi-mandibles (n=10 per time point, 5 control unoperated and 5 implanted) were scanned using a Microcomputed Tomography (μCT) imaging system (Skyscan 1176, Bruker MicroCT, Billerica, MA). Scans were performed on all harvested implants using the set parameters of 100 kV, 100 A, Al-Cu filter, 0.3 rotation step over 180° and pixel size 9 m, together with two BMD phantoms with BMD values of 0.25 and 0.75 g / cm-3. μCT data was then reconstructed to rebuild acquisition datasets using NRecon software (Bruker MicroCT). The region of interest (ROI), defined as a full thickness (6 mm), 10 mm diameter circle that matched the defect area was further selected and evaluated for new bone regeneration using Avizo (Version 1.6.9.15, ThermoFisher Scientific, Materials & Structural Analysis Division, Hillsboro, Oregon) and CTAn (Bruker MicroCT) software. A full thickness 10 mm diameter, 6 mm wide region on the unoperated control right side mandible was similarly analyzed (Supplement figure 1). Harvested hemi-mandibles were then decalcified in (1:1) 45% Formic Acid:20% Sodium Citrate solution for one month. The Ti dental implants were carefully removed for subsequent SEM analyses, and the remaining bone implants were prepared for paraffin embedding and sectioning, and histological / IF analyses. Paraffin sections were analyzed using Hematoxylin and Eosin (H&E) and Masson’s Trichrome staining. Immunofluorescent (IF) staining was performed using primary antibodies for the odontoblast differentiation marker Dentin Sialophosphoprotein (DSPP, abx176139, Abbexa Ltd, Cambridge, United Kingdom), and - smooth muscle actin (SMA, Ab21027, Abcam, Cambridge, United Kingdom) for blood vessel formation, and anti- human MHC classHLA B antibody (Ab134189, Abcam) to detect any human DPSCs / HUVECs in the implants. To evaluate hDPSCs and HUVEC distribution throughout TyroFill scaffolds prior to implantation, replicate (2) cell-seeded constructs were embedded in OCT, cryosectioned and subjected to histological and IF analyses. IF staining was performed on replicate (5) sections that spanned each scaffold using the primary antibodies for the mesenchymal cell marker Vimentin (VM, sc-6260, Santa Cruz Biotechnology, Dallas, TX) and the endothelial cell marker Factor VIII (ab61910, Abcam), and appropriate secondary antibodies. Replicate (3) 40x images were taken on each section by an M2-Bio Zeiss fluorescent microscope(Zeiss, Germany). Positive cells were identified and quantified via Image J software (National Institutes of Health, Bethesda, MD, USA).

[0049] Surface characterization of the titanium dental implants

[0050] The surface morphology and elemental composition of un-implanted control and implanted Ti dental implants (n=10) were analyzed via Scanning Electron Microscope (SEM) and Energy- Dispersive X-Ray (EDAX), respectively. Ti dental implants were dehydrated in graded ethanol series and HMDS (Hexamethyldisilazane), sputter coat by gold / palladium, and analyzed by SEM (Hitachi S-4800, Tokyo, Japan) at the Northeastern University EM Facility (Boston, MA). Qualitative chemical composition at three different sites on each Ti implant was assessed by EDAX. Two non-implanted starting material Ti dental implants were processed and analyzed as controls.

[0051] Results

[0052] Construction and in vitro culture of 3D bone-tooth constructs.

[0053] FIGs. 2A-2F illustrate characteristics of TyroFill-Ti constructs. hDPSCs) (FIG. 2A) and HUVECs (FIG. 2B) showed typical morphologies prior to cell seeding. In vitro expanded hDPSCs and HUVECs exhibited a healthy appearance prior to seeding onto scaffolds, as shown in FIG. 2A. TyroFill scaffold fabrication was performed as previously published. No obvious changes were observed in cell-seeded or acellular TyroFill scaffolds after one-week in vitro culture in osteogenic media, as shown in FIG. 2B. The inserted dental implant remained stable prior to implantation, as shown in FIG. 2C, which shows the TyroFill-Ti constructs after 1-week in vitro culture in OM, with cells (upper panel) or without cells (lower panel). D0 indicates scaffolds immediately after cell-seeding, and D7 indicates scaffolds immediately prior to implantation. Histological analyses of cryosectioned cell-seeded constructs prior to implantation stained with H&E revealed good cell attachment and morphology throughout the constructs, as shown in FIG.2D, which shows abundant cell distribution throughout the TyroFill construct before implantation. The arrows of FIG. 2D arrows indicate some cell clusters. Immunostaining of mesenchymal cell marker Vimentin and endothelial cell marker Factor VIII revealed that hDPCs and HUVECs remained at an approximate 1:1 ratio after 1 week culture in osteogenic media, as shown in Figures 2E and 2F. As shown in FIG. 2E, double IF staining revealed both hDPSCs 210 and HUVECs 212 in Tyrofill implant prior to implantation. As demonstrated in FIG. 2F, statistical analysis showed that hDPSCs and HUVEC retained an ~1:1 ratio prior to implantation. Abbreviations: Ti, Titanium implant; VM, Vimentin; Fac VII, factor 8. Scale bars: (FIGs.2B and 2C) 1 mm; (FIGs. 2A and 2D) 100 μm; (FIG.2F) 20 μm.

[0054] Post-surgical analyses.

[0055] All rabbits showed excellent recovery and healing after surgery, and no weight loss or other adverse reactions were observed. After 1 and 3 months, no noticeable changes in the dentition or jawbone were observed in any of the implanted jaws as compared to the contralateral unoperated control mandible. As shown in FIG. 3A, three dimensional (3-D) μCT analyses of the harvested jaws with implants showed an easily identifiable radiolucent circular defect site and highly radiopaque dental implant in all harvested mandibles at 1 and 3 months. Radiopaque areas at the implant site indicated newly formed bioengineered mineralized tissue. Comparatively more mineralized tissue formation was observed at 3 months as compared to 1- month post-implantation. FIGs. 3A-3B show microcomputed tomography (μCT) analyses of harvested implants. As can further be seen in FIG. 3A, hDPSC-HUVEC cell seeded implants exhibited more uniform calcified tissue formation throughout the entire implant site as compared to implanted acellular constructs. FIG. 3A (scale bars: (A) 2mm) shows representative 3-D μCT images of harvested 1-month (upper panel) and 3-month (lower panel) implants. A full thickness 10 mm diameter cylindrical area 310 that matched the defect area was selected, and the Ti implant was excluded based on its distinctive density 312. Increased amounts of radiopaque calcified tissue were observed in 3-month as compared to 1-month implanted constructs. hDPSCs / HUVEC-seeded TyroFill constructs exhibited more homogeneous mineralized tissue formation throughout the implants as compared to acellular constructs. As shown in FIG.3B, new hard tissue formation was quantified for bone density, bone volume / tissue volume (BV / TV) measurements within the selected defect site area using μCT image analyses. Trabecular thickness and distribution were quantified to evaluate the maturity of newly formed bone. Both measurements showed that cell-seeded constructs exhibited increased bone volume and maturity over time as compared to acellular construct implants although no significant difference was observed due to the limited number of implants. Comparatively, TyroFill constructs showed more robust new bone formation as compared to previously characterized E1001(1K)-bTCP scaffolds implanted using similar conditions. FIG. 3B shows the quantification of new bone volume / tissue volume (BV / TV) and trabecular thickness within the constructs at the implant site. Cell seeded constructs at later timepoints showed greater and more mature bone formation. Error bars indicate the standard deviation among samples.

[0056] Histological analyses of bioengineered constructs.

[0057] Turning now to FIGs. 4A-4D, histological analyses of harvested TyroFill implants are shown. H&E staining of coronally sectioned harvested constructs was used to assess bioengineered bone formation at the defect site. Histological analyses showed that most TyroFill scaffold pores were filled with well-organized soft tissues in 1-month cell seeded samples, while1-month acellular samples exhibited reduced soft tissue density and volume. Bioengineered bone fragments similar to that of natural bone were detected in cell-seeded 1-month implants, as demonstrated in FIG.4A, which shows H&E stained 1-month hDPSCs / HUVEC seeded. FIG. 3B shows acellular TyroFill constructs. After 3 months implantation, cell-seeded TyroFill constructs showed robust bone formation throughout the entire defect area, as demonstrated in FIG. 4C, which shows 3-month hDPSCs / HUVEC seeded constructs. In FIGs. 4A-4D, panels 1 and 2 show high magnification images of boxed areas in A, B, C, and D. The void left by the removed Ti implant is outlined as shown. After 3 months implantation, cell-seeded constructs showed new bone formation throughout (see C1, C2 of FIG. 4C), while no obvious bone formation was evident in acellular constructs (see D1, D2 or FIG. 4D). Although new bone formation was observed around the periphery of the implant area, no obvious bone formation was present near the center of acellular constructs, as demonstrated in FIG. 4D. TheE1001(1k), the polymer used in the manufacture of the TyroFill scaffold appeared green in 1-month implants when viewed using a fluorescent GFP filter, particularly in acellular implants, as shown in the GFP panel, indicated by the arrows, in FIGs. 4A and 4B. As shown in FIGs. 4C-4D, no noticeable polymer was observed in TyroFill constructs after 3 months implantation. A’-D’ in FIGs. 4A-4D, respectively, show the remaining scaffold using GFP filter, where the arrows indicate undegraded TyroFill. Corresponding polarized light images (A”-D”) confirm new bone formation, where the arrows indicate areas of mature new bone formation. Abbreviations: M, Mandible; T, Tooth. Scale bar = 1 mm (A-D), 50 μm (all other panels).

[0058] Immunofluorescent staining performed using bone / dentin markers also indicated robust bioengineered alveolar bone formation, particularly in DPSC / HUVEC-seeded Tyrofill constructs. FIGs. 5A-5B illustrate immunofluorescent analyses of bone differentiation marker expression in bioengineered bone TyroFill constructs, where the arrows 510, 512 indicate human (MHC+) cells expressing DSPP or -SMA. Representative images were taken of sections obtained from the center of harvested implants. Positive DSPP expression was observed in all harvested implants at 1 and 3 months, with stronger expression observed in cell-seeded constructs especially after 3-months hDPSCs / HUVEC seeded implantation (see arrows 510 of FIG. 5A). Strong -SMA expression (see arrows 510 of FIG. 5B) indicated blood vessel formation throughout the implants, including in the center of the implants, especially in 3-month harvested cell-seeded implants. MHC positive human DPSCs / HUVECs, indicated by the arrows 512 of FIG. 5B, were only detected in 1-month hDPSCs / HUVEC seeded TyroFill constructs. The scale bar of FIGs.5A-5B is 50 μm.

[0059] Characterization of harvested dental implant surfaces

[0060] FIGs. 6A-6B demonstrate analyses of Ti implant surfaces removed from harvested TyroFill constructs, indicating calcified matrix deposition on the implant surface. Referring to FIGs. 6A, SEM analyses of un-implanted Straumann Ti dental implants showed an irregular honeycomb structure similar to that of previously published reports. No significant difference was observed on the surface untreated and 1 week in vitro cultured dental implants (see FIG. 6A). The arrows 310 of FIG. 6A indicate areas containing cells, and the arrows 320 indicate calcified nodule formation.

[0061] After in vivo implantation at 1 and 3 months, the surface of all acellular and cell- seeded Ti implants exhibited deposition of highly organized extracellular matrix (ECM). Implants removed from the cell-seeded constructs exhibited clearly identifiable cells on the surface (2000x) (see FIG. 6A). Relatively increased calcified nodule formation was observed on the surface of implants retrieved from 3-month cell-seeded constructs (see FIG. 6C) as compared to 1 month (see FIG. 6B) implanted constructs. As expected, Energy-Dispersive X-Ray (EDAX) analyses revealed the presence of titanium on the surface of all Straumann Ti dental implants (see FIGs. 6B-6C). EDAX analysis also confirmed surface deposition of bone matrix containing higher levels of Ca in cell-seeded constructs (see FIGs. 6B-6C). No significant difference was found between cell-seeded and acellular samples, likely due to insufficient sample numbers. The elemental spectral peaks include significant amounts of Titanium and Calcium, as indicated.

[0062] Discussion

[0063] Successful therapies to regenerate bone in large, critical sized CMF defects require bone grafts that exhibit high biocompatibility, sufficient mechanical properties to support CMF structure and masticatory function during the bone healing process, and the ability to be easily handled and accurately shaped to precisely fit the uniquely complex anatomies of craniomaxillofacial bones. Ideally, these scaffolding materials should also be biodegradable, and eventually be replaced by robust and vital newly formed bone. Autogenous bone is considered the current gold standard, and autogenous cryogenically preserved autogenous extracted teeth have also been proposed as another source of natural mineralized tissue for tissue regeneration.

[0064] Although the mechanisms regulating carbonate hydroxyapatite (CAP)-induced bone formation are still incompletely understood, the superior osteoinductive properties of CAP makes it one of the best scaffolds for bone regeneration. For example, one study used CAP granules combined with stem cells from human exfoliated teeth (SHEDs), transplanted to the defect using an atelocollagen sponge scaffold, to treat calvarial defects in immunocompromised 6-week-old male immunodeficient mice. Analyses of these harvested implants clearly showed that the SHEDs + CAP transplantation group exhibited significantly higher bone regeneration ascompared to the CAP alone and SHEDs alone groups. Ideally, the most promising scaffolds would consist of biomimetic organic scaffolds coated with CaP to mimic the organic-inorganic composition of native bone. Previously published results showed that E1001(1k) derived porous scaffolds supported robust bone regeneration in a rabbit critical-sized calvarial defect, and in an ovine long bone defect repair model. Previously published results also demonstrated that E1001(1K)-bTCP scaffolds are very effective for mandibular jaw regeneration in a critical- sized rabbit mandible defect model. Based on these promising results, and the fact that a new E1001(1k) formulation, TyroFill, also effectively supports in vitro cultured DPSC proliferation and differentiation, the objective of this study was to test whether hDPSC / HUVEC-seeded TyroFill scaffolds were effective in repairing alveolar bone in an in vivo rabbit mandibular defect repair model. The TyroFill scaffolds used in this study exhibited bimodal, interconnected macro and micro porous structure with > 90% final porosity that mimicked the pore size range and architecture of trabecular bone.

[0065] The results showed mineralized tissue regeneration within and on the surface of both hDPSC / HUVEC seeded and acellular TyroFill scaffolds, in accordance with the highly porous and osteoconductive nature of these scaffolds. The presence of new bone formation within and on the surface of acellular Tyrofill scaffold implants indicates the ability of TyroFill scaffolds to recruit host cell participation in mineralized tissue regeneration. μCT and histological analyses showed that rabbit mandibles implanted with hDPSC / HUVEC-seeded TyroFill scaffolds exhibited a unique pattern of mineralized tissue formation as compared to acellular scaffolds. Fewer but larger areas of calcified tissue formed largely in the periphery and not the center of acellular scaffold implants, while smaller areas of homogeneous and evenly distributed new bone formed throughout hDPSC / HUVEC seeded constructs. Furthermore, the expression of DSPP, a dentin-specific matrix protein also expressed in naturally formed alveolar bone, was only observed in bioengineered bone derived from hDPSC / HUVEC-seeded TyroFill constructs and not in acellular constructs. Together, these results suggest that TyroFill scaffolds exhibit the ability to support hDPSC differentiation, vascularized tissue formation, and the formation of mineralized tissue resembling that of natural jawbone. It was further demonstrated that MHC expressing hDPSC / HUVECs were detectable in cell seeded TyroFill implants harvested at 1 month, but not at 3 months. These results are consistent with numerous reports showing that implanted human cells contribute to long term tissue regeneration, but do not maintain long term residence in the implants.

[0066] In natural bone formation and remodeling, osteogenesis and angiogenesis are tightly coupled processes. Blood vessels not only carry oxygen and nutrients to developing bone, butalso play an active role in mediating interactions between osteoblasts, osteocytes, osteoclasts and endothelial cells. A unique property of E1001(1k) derived scaffolds is their highly organized micro-architecture consisting of a highly interconnected porosity that facilitates efficient cell infiltration through macropores (200 - 400 m), and efficient delivery of nutrients throughmicropores (< 20 m). For the study described here, 10 mm diameter x 6 mm highcylindrical TyroFill scaffolds were seeded with both HUVECs and hDPSCs, to facilitateangiogenesis and alveolar bone formation, respectively. In fact, the results showed significant blood vessel formation throughout the cell-seeded implanted constructs, especially after 3- months implantation.

[0067] With respect to facilitating the osseointegration of Ti implants, a porous scaffold could increase the long-term mechanical stability by facilitating bone growth into the highly porous scaffold and around the Ti implant. It was previously shown that a pore size range of 100 – 600 m could promote efficient osseointegration. The TyroFill scaffold with Ti implant used in this study exhibited similar pore size, which efficiently promoted calcified tissue formation throughout the TyroFill scaffold and around the Ti implant, particularly in cell-seeded constructs.

[0068] In summary, the studies described here demonstrate the potential for hDPSCs- HUVEC seeded TyroFill constructs as a potential new and improved therapy to efficiently repair CMF defects. TyroFill constructs exhibited better bone forming capability as compared topreviously used hDPSC-seeded E1001(1k) / -TCP scaffolds indicating the importance of theDCDP coating for bone regeneration. The observed, robust new bone formation within TyroFill scaffolds and on the surface of Ti implants, especially in cell-seeded constructs, indicates the potential utility of TyroFill-Ti scaffolds as a potentially new and more effective therapy for coordinated CMF bone and tooth regeneration, to improve patient outcomes and reduce surgical costs.

[0069] All animal experiments were conducted under the guidance and approval of the Institutional Animal Care and Use Committee (IACUC) of Tufts University (protocol number: B2020-01).

[0070] The experiments described herein were conducted with different biodegradable polymers for manufacturing the scaffolds. The main selection criteria included degradability within the body of the patient within about 1 year, biocompatibility, and the release of degradation products that are non-toxic and non-inflammatory to the surrounding tissue.

[0071] These criteria are met by several classes of known biodegradable medical polymers, including, but not limited to, copolymers of lactic acid and glycolic acid, poly(lactide glycolides) abbreviated as PLGA, and members of the family of tyrosine-derived polycarbonates. Thefamily of tyrosine-derived polycarbonates comprises hundreds of individual polymers whose specific compositions are identified by a unique 6-letter code. Accordingly, one of the preferred embodiments is E1001(1k), a copolymer of 89 mole% of desaminotyrosyl-tyrosine ethyl ester, 10 mole% of desaminotyrosyl-tyrosine, and 1 mole% of poly(ethylene glycol) having a molecular weight of 2 kDa.

[0072] Each of the above embodiments and obvious variations thereof are contemplated as falling within the spirit and scope of the claimed invention, which is set forth in the following claims. Moreover, the present concepts expressly include any and all combinations and sub- combinations of the preceding elements and aspects.

[0073] As utilized herein, the terms “approximately,” “about,” “substantially,” and similar terms are intended to have a broad meaning in harmony with the common and accepted usage by those of ordinary skill in the art to which the subject matter of this disclosure pertains. It should be understood by those of skill in the art who review this disclosure that these terms are intended to allow a description of certain features described and claimed without restricting the scope of these features to the precise numerical ranges provided. Accordingly, these terms should be interpreted as indicating that insubstantial or inconsequential modifications or alterations of the subject matter described and claimed are considered to be within the scope of the invention as recited in the appended claims.

[0074] It should be noted that the terms “exemplary” and “example” as used herein to describe various embodiments are intended to indicate that such embodiments are possible examples, representations, and / or illustrations of possible embodiments (and such terms are not intended to connote that such embodiments are necessarily extraordinary or superlative examples).

[0075] Although only a few embodiments have been described in detail in this disclosure, those skilled in the art who review this disclosure will readily appreciate that many modifications are possible (e.g., variations in sizes, dimensions, structures, shapes and proportions of the various elements, values of parameters, mounting arrangements, use of materials, colors, orientations, etc.) without materially departing from the novel teachings and advantages of the subject matter described herein. For example, elements shown as integrally formed may be constructed of multiple parts or elements, the position of elements may be reversed or otherwise varied, and the nature or number of discrete elements or positions may be altered or varied. The order or sequence of any process or method steps may be varied or re-sequenced according to alternative embodiments. Other substitutions, modifications, changes, and omissions may alsobe made in the design, operating conditions, and arrangement of the various exemplary embodiments without departing from the scope of the present invention.

Claims

What Is Claimed Is:

1. A composition for use in treating a dental implant, the composition comprising: a porous biodegradable polymer scaffold; and a dicalcium phosphate dihydrate (DCPD) coating within pores of the scaffold.

2. The composition of claim 1, wherein the biodegradable polymer scaffold was prepared by porogen leaching.

3. The composition of claim 1, further comprising cells seeded within the scaffold, the cells including human dental pulp stem cells and human umbilical vein endothelial cells 4. The composition of claim 3, wherein the ratio of human dental pulp stem cells to human umbilical vein endothelial cells is approximately 1:

1.

5. The composition of claim 3, wherein the human dental pulp stem cells have been isolated from human dental pulp.

6. The composition of claim 5, wherein the dental pulp is from a mature tooth.

7. The composition of claim 6, wherein the mature tooth is a wisdom tooth.

8. The composition of claim 3, wherein the composition has a density of about 0.25 x 105cells / mm3.

9. The composition of claim 1, wherein the scaffold includes a bimodal, interconnected macro and micro porous structure with about 70% to about 95% final porosity. 10 The composition of claim 1, wherein the scaffold includes pores having a size range of about 100 μm to about 600 μm.

11. The composition of claim 1, wherein the biodegradable polymer is a tyrosine-derived polycarbonate 12. The composition of claim 11, wherein the tyrosine-derived polycarbonate is a composition designated as E1001(1k).

13. A method of forming a composition for use in treating a dental implant, the method comprising: coating the pores of an E1001(1K) scaffold with dicalcium phosphate dihydrate (DCPD) to form a E1001(1K) / dicalcium phosphate dihydrate (DCPD) scaffold. 1448.87-8203-3 T543h.1e method of claim 13, further comprising forming the E1001(1K) scaffold by 700355-000100USPL porogen leaching.

15. The method of claim 13, further comprising seeding human dental pulp stem cells and human umbilical vein endothelial cells in the E1001(1K) / dicalcium phosphate dihydrate (DCPD) scaffold.

16. The method of claim 15, further comprising pretreating the E1001(1K) / dicalcium phosphate dihydrate (DCPD) scaffold in mesenchymal cell Osteogenic Media prior to the seeding to develop a calcium coating.

17. The method of claim 15, wherein the ratio of human dental pulp stem cells to human umbilical vein endothelial cells is approximately 1:

1.

18. The method of claim 15, wherein the human dental pulp stem cells have been isolated from human dental pulp.

19. The method of claim 13, wherein the scaffold includes a bimodal, interconnected macro and micro porous structure with about 70% to about 95% final porosity.

20. The method of claim 13, wherein the scaffold includes pores having a size range of about 100 μm to about 600 μm.

21. A method of a treating dental implant, the method comprising: immersing at least a portion of the implant into a scaffold comprising E100(1K) / dicalcium phosphate dihydrate (DCPD).

22. The method of claim 21, wherein the scaffold is seeded with human dental pulp stem cells, human umbilical vein endothelial cells, or a combination thereof.

23. The method of claim 22, wherein the scaffold has a density of about 0.25 x 105cells / mm3.

24. The method of claim 21, wherein the dental implant comprises titanium.

25. The method of claim 21, wherein the immersing results in a coating of dicalcium phosphate dihydrate being formed on a surface of the dental implant.

26. The method of claim 21, wherein the duration of the immersing is from about 1 day to about 7 days. 4887-8203-3543.1 700355-000100USPL

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