Oxidation-responsive resins

Abstract

A short-chain poly(thioketal) ("PTK") polymer is provided. The polymer is produced by a method comprising reacting 2,2 dimethoxypropane (DMP) or a ketone with a dithiol monomer in the presence of an acid using a nitrogen atmosphere and stirring for a period of time from 1 to 72hrs. The ketone is selected from the group consisting of acetone, levulinic acid, pyruvic acid, sulfonyl acetone, and oxoglutaric acid.

Description

OXIDATION-RESPONSIVE RESINSCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional Application Serial No. 63 / 553,127, filed February 13, 2024, which application is hereby incorporated by reference in its entirety.FIELD OF THE INVENTION

[0002] This invention relates generally to photopolymerizable resins.BACKGROUND OF THE INVENTION

[0003] This section is intended to introduce the reader to various aspects of art that may be related to various aspects of the present invention, which are described and / or claimed below. This discussion is believed to be helpful in providing the reader with background information to facilitate a better understanding of various aspects of the present invention. Accordingly, it should be understood that these statements are to be read in this light, and not as admissions of prior art.

[0004] Environmentally responsive polymers that can tie programmed material functionality to local biological stimuli have garnered increasing interest in tissue engineering research. However, currently demonstrated polymer systems that are triggerable by biological stimuli such as cell-generated reactive oxygen species (ROS) are significantly limited by two factors: they have not achieved rapid responsivity to physiological doses of ROS, and they have not been adapted for use with advanced material fabrication techniques such as 3D printing. Therefore, a need still exists for new responsive biomaterials possessing increased sensitivity to ROS and compatibility with additive manufacturing.SUMMARY OF THE INVENTION

[0005] Certain exemplary aspects of the invention are set forth below. It should be understood that these aspects are presented merely to provide the reader with a brief summary of certain forms the invention might take and that these aspects are not intended to limit the scope of the invention. Indeed, the invention may encompass a variety of aspects that may not be explicitly set forth below.

[0006] In one aspect of the present invention, a short-chain poly(thioketal) (“PTK”) polymer is provided. The polymer is produced by a method comprising reacting 2,2 dimethoxypropane (DMP) or a ketone with a dithiol monomer in the presence of an acid using a nitrogenatmosphere and stirring for a period of time from 1 to 72hrs. The ketone is selected from the group consisting of acetone, levulinic acid, pyruvic acid, sulfonyl acetone, and oxoglutaric acid. In one embodiment, the dithiol monomer is selected from the group consisting of 2- mercapto ethyl ether (MEE) and 3,6-dioxa 1,8 -octanedi thiol (DOT). In one embodiment, a bone cement for orthopedic implant fixation is provided. The bone cement has a composite of the short-chain PTK polymer described above and calcium phosphate (“CaP”). In another embodiment, the composite includes short-chain PTK polymer, a multi-functional epoxy linker, and hydroxyapatite (HAp) particles. In one embodiment, the multi-functional epoxy linker comprises trimethylolpropane triglycidyl ether (“TA”). In another embodiment, the composite further includes choline hydroxide. In one embodiment, the HAp particles have an average diameter of 50pm.

[0007] In another aspect of the present invention, a three-dimensional (“3D”) printable resin is provided. The resin includes a composite of the short-chain PTK polymer described above and a multi-functional linker possessing photopolymerization capabilities. In one embodiment, the tetra-functional linker is selected from the group consisting of trimethylolpropane triallyl ether (AE) and trimethylolpropane trimethacrylate (TMA). In another embodiment, the composite further includes Irgacure 819 (“BAPO”). In another embodiment, the composite further includes 2,2,6,6-tetramethylpiperidinyl-l-oxyl (“TEMPO”).

[0008] In another aspect of the present invention, a method of testing a 3D printed construct for treatment of a bone defect is provided. The method involves implanting a 3D printed construct comprising the 3D printable resin described above into a rat calvarial bone defect and evaluating response factors comprising bone growth kinetics, implant degradation timelines, and local tissue response.

[0009] In another aspect of the present invention, a method of testing a construct for treatment of a bone defect is provided. The method involves implanting a construct comprising the bone cement described above into a rat calvarial bone defect and evaluating response factors comprising bone growth kinetics, implant degradation timelines, and local tissue response.BRIEF DESCRIPTION OF THE DRAWINGS

[0010] The objects and advantages of the disclosed invention will be further appreciated in light of the following detailed descriptions and drawings in which:

[0011] FIG. 1 is a schematic showing a summary of one embodiment of the present invention, SC-PTK is utilized in two TEC systems as a bone cement for orthopedic implant fixation andas a 3D printable construct amenable to SLA. The intention is to introduce ROS degradable materials as alternatives to conventionally used biomedical polymers.

[0012] FIG. 2 is a graph showing gel permeation chromatography (GPC) chromatogram demonstrating elution times of varying synthesis protocols for fabrication of SC-PTK. The graph shows an unoptimized PTK, non-heated PTK, and the final optimized SC-PTK.

[0013] FIG. 3A is a graph showing compressive modulus measurement of cement scaffolds with varying HAp.

[0014] FIG. 3B is a graph showing yield strain modulation as a function of HAp.

[0015] FIG. 3C is a graph showing cement time-dependent properties with PBS incubated samples.

[0016] FIG. 3D is a graph showing selective ROS degradation in H2O2 media and nondegradation in aqueous media.

[0017] FIG. 4A is a graph showing tac free time of varying catalyst cement formulations.

[0018] FIG. 4B is a graph showing LD50 done in choline conditioned media.

[0019] FIG. 4C is a graph showing temperature profile of curing cement after initial homogenization.

[0020] FIG. 4D is a graph showing cell viability of MC3T3-E1 cells in conditioned PTK bone cement media.

[0021] FIG. 5 A is a photo of cylindrical PTK bone cement removed from mold for characterization.

[0022] FIG 5B is an SEM of PTK bone cement microstructure.

[0023] FIG. 6A is an image showing SLA resins created with TK dithiols (A) are miscible with -ene crosslinkers,

[0024] FIG. 6B is an image showing that the SLA resins can be 3D printed into small-geometry shapes.

[0025] FIG. 6C is a graph showing samples printed from the SLA resins selectively degrade when treated with ROS.

[0026] FIG. 7 is a schematic showing a method of creating a cell-degradable implant.

[0027] FIG. 8 is a schematic showing resin precursors that participate in a thiol-ene polymerization.

[0028] FIG. 9 is a graph showing -ene terminated reagent stability in UV.

[0029] FIG. 10 is a graph showing FTIR being used to determine thiol elimination in UV and non-UV exposed samples.

[0030] FIG. 11 A is a graph showing data from conducted cured depth experiments.

[0031] FIG. 1 IB is a graph showing data from conducted cured depth experiments.

[0032] FIG. 11C is a graph showing data from conducted cured depth experiments.

[0033] FIG. 12A is a pair of images showing a benchmark structure print for TK-TMA.

[0034] FIG. 12B is a pair of images showing a benchmark structure print for TK-AE.

[0035] FIG. 13 A is a graph showing degradation data for a TK-TMA formulation.

[0036] FIG. 13B is an image showing printed dog bone samples for performing tensile tests and measuring mechanical properties of each resin formulation.

[0037] FIG. 14A is a graph showing TK-AE rheometry cured depth.

[0038] FIG. 14B is a graph showing TK-TMA IB 0.2T cured depth.

[0039] FIG. 14C is a graph showing TK-AE IB 0.2T combined cured depth.

[0040] FIG. 14D is a graph showing cumulative cures.

[0041] FIG. 14E is a graph showing cured depth studies TK-TMA 1.

[0042] FIG. 14F is a graph showing cured depth studies TK-TMA 1.DEFINITIONS

[0043] As used herein, the term “about,” when referring to a value or to an amount of mass, weight, time, volume, pH, size, concentration or percentage is meant to encompass variations of ±20% in some embodiments, ±10% in some embodiments, ±5% in some embodiments, ±1% in some embodiments, ±0.5% in some embodiments, and ±0.1% in some embodiments from the specified amount, as such variations are appropriate to perform the disclosed method.DETAILED DESCRIPTION OF THE INVENTION

[0044] One or more specific embodiments of the present invention will be described below. In an effort to provide a concise description of these embodiments, all features of an actual implementation may not be described in the specification. It should be appreciated that in the development of any such actual implementation, as in any engineering or design project, numerous implementation-specific decisions must be made to achieve the developers’ specific goals, such as compliance with system-related and business-related constraints, which may vary from one implementation to another. Moreover, it should be appreciated that such a development effort might be complex and time consuming, but would nevertheless be a routine undertaking of design, fabrication, and manufacture for those of ordinary skill having the benefit of this disclosure.

[0045] Poly(thioketal) (PTK) polymers are an emerging class of materials with exciting potential applications within the biomedical domain. PTKs are synthetic biomaterials that arespecifically degraded by oxidative molecules such as the cell-produced signal reactive oxygen species (ROS). Current dogma suggests that ROS are implicated in tissue regeneration mechanisms and are involved in immunologic cell signaling. Moreover, this is especially true following skeletal fracture for complete osteogenesis. By possessing an innate ability to degrade selectively in the body due to ROS, PTK polymers can achieve bio-responsiveness at the cellular level driving material degradation. This is advantageous within tissue engineering thrusts to utilize PTK-based macromolecules as a tissue engineering construct (TEC). The present invention leverages the presence of ROS native to the bone healing process to directly tie material degradation to tissue regeneration; thus, achieving complete biodegradation of implanted PTK polymers, improving incorporation of native tissue to an orthopedic implant, and improving patient prognosis post orthopedic implant surgery.

[0046] In one embodiment, the present invention involves the use of PTK materials’ bioresponsiveness for the development of PTK bone cements for orthopedic implant fixation. In a second embodiment, the present invention involves the use of PTK materials’ bioresponsiveness for the creation of a PTK-based resin amenable to stereolithography (SLA) 3D printing. In a third embodiment, the present invention involves the use of PTK materials’ bioresponsiveness for performance evaluation of these PTK TECs in vivo using a critically sized rat calvarial defect model.Development of a PTK bone cement for orthopedic implant fixation

[0047] Traditional bone cements, most notably calcium phosphates (CaP) and poly(methyl methacrylate) (PMMA) formulations, have been employed clinically as a fixation material for several types of artificial joint replacements. However, these traditional cements possess innate caveats: PMMA is non-resorbable and does not foster strong anchoring or tissue in-growth with native bone, often leading to fibrosis at the bone-implant interface, increasing the inflammatory response around the implant area that causes pain, poor healing outcomes, and an increased potential for further medical complications. Conversely, pure CaP cement better integrates with native bone tissue but suffers from brittle mechanical properties that are not equipped to withstand forces associated with weight bearing implants. The present invention has found that PTK-based CaP composite bone cement can address issues observed with both PMMA and pure CaP cement formulations. PTK polymer containing reactive dithiol end- groups can be combined with a tri-epoxy linker, hydroxyapatite (HAp) particles, and a nontoxic quantity of choline catalyst to create an ROS-degradable fixation material. In vitro testing can demonstrate selective oxidative degradation, robust tunable mechanical properties, limitedcytotoxicity, and non-thermogenicity with these materials. This new class of orthopedic implant fixation material rivals current clinically utilized alternatives.3D printable SLA resin using TK monomers

[0048] Within the field of tissue engineering, additive manufacturing (AM) techniques allow for the design, creation, and implementation of tissue engineering constructs (“TECs”) with complex morphologies. SLA is an AM technique which leverages UV-induced polymerization of a liquid resin by irradiation of light in a layer-by-layer manner to create 3D morphologies. Furthermore, SLA allows for precise control of TEC internal structure allowing for precise pore sizes, and pore geometries allowing greater cellular infiltration. The present invention has found that this technology can be leveraged to construct scaffold that mimic native tissue. TK chemistries have not previously been utilized in 3D printable applications. Therefore, a resin mixture can be created incorporating TK monomers and a tetra-functional linker possessing photopolymerization capabilities. The mechanical properties, curing profile, selective ROS degradation, amenability to SLA 3D printing, and in vitro cytocompatibility of the novel TK based resin mixture of the present invention can be characterized, demonstrating the efficacy of this bio-responsive resin for biomedical prototyping and implantation.The effects of PTK containing TECs within a critically sized rat calvarial model

[0049] Bone possesses a unique ability to regenerate to near full function after fracture. However, when a bone fracture is too large, the fracture is deemed “critically sized” and features distinctly poor bone regeneration outcomes that require additional intervention. To evaluate the performance of PTK TECs in a challenging in vivo environment, both PTK-based cements and 3D printed constructs can be implanted into critically sized rat calvarial bone defects. Then bone growth kinetics, implant degradation timelines, and the local tissue response can be evaluated. The PTK bone cement of the present invention can be benchmarked to traditional PMMA, hydrolytically degradable cement analogues, and a negative control. Furthermore, the SLA-printed PTK implants of the present invention can be compared to a non-ROS degradable alternative. Implant outcomes can be characterized via immunohistochemistry, micro-CT, and histological staining.Treatment of bone defects

[0050] The field of tissue engineering (TE) currently seeks to augment tissue regeneration and functional outcomes post injury or during a disease state, where an acceptable regenerativeconclusion is not ordinarily observed. TE aims to provide reproducible clinical outcomes in a plethora of disease conditions, tissue architectures, and surgical procedures where loss of tissue is observed or required. The translatability of solutions to the clinic is highly variable when considering which specific tissue type or disease state is being addressed. Successful solutions are observed through alterations of material and chemical properties, presentation of regenerative cues, biomechanical cell-matrix interactions, 3D printing of tissue specific constructs, and many more careful considerations. Regenerative outcomes for bone injuries are typically positive for most individuals suffering from fractures, however, misunion of defects, slow regenerative timescales, or no observable regeneration are some prognoses which demand TE solutions. Specifically, for osseus defects that are deemed as ‘critically sized’, spontaneous regeneration is not observed, and corrective strategies must be employed.

[0051] There is much debate on what specifically constitutes a critically sized bone defect. Defect size can vary by anatomic position and by person. The current consensus of treatment for critically sized bone defects is handled through individualized treatment and adherence to principles of care when observed in the clinic. One of the clinically induced procedures which necessitates removal of native bone is during artificial joint replacement (AJR) surgeries. Typically, AJR is utilized as a solution to wear and tear observed at a joint interface, and survival rates are >90%. For certain AJR procedures, exogenous fixation materials are utilized to prolong implant function and are an integral part of orthopedic surgical procedures. The current gold standard for orthopedic implant fixations are typically derived from poly(methyl methacrylate) (PMMA) and calcium phosphate (CaP) formulations.

[0052] PMMA and CaP bone cements have been demonstrated to enhance stability in hip fracture fixation, stabilization of osteoporotic femurs, and are utilized in high to medium load bearing applications. However, acrylic bone cement possesses innate caveats as the material cannot be remodeled when in the body, the exothermic reaction of the cement can hinder fracture regeneration, and the high elastic modulus (1-2 GPa) can result in stress shielding and implant loosening that can lead to implant failure. Additionally, PMMA is comprised of liquid precursors that undergo live polymerization to fixate implants. This process is exothermic reaching temperatures up to 83.48 ± 7.35 °C, inducing tissue necrosis, and poor healing outcomes. There are ongoing efforts to ameliorate exothermicity of PMMA CaP bone cements. A common focus in TE is centered around the development of alternative materials that address pitfalls of currently utilized AJR fixation materials.

[0053] One strategy employed herein leverages the endogenous, cell produced signals that are present during bone regrowth, reactive oxygen species (ROS). ROS acts both as signalingmolecules and mediators of inflammatory pathways within the local injury environment. Specifically, ROS induces migration and proliferation of endothelial cells and secretion of vascular endothelial growth factor (VEGF) which are imperative for complete osteogenesis. Conversely, at high concentrations of ROS beyond physiologically relevant levels, onset of oxidative stress is observed. Persistence contributes to disease pathogenesis and cellular damage hindering fracture healing outcomes.

[0054] An emerging class of materials derived from ROS sensitive polymeric backbones are being developed for drug delivery, regenerative medicine, and TE applications. Polymeric structures that are stable in aqueous conditions and undergo structural modification in the presence of localized ROS have shown promise in the biomedical realm. Due to great material stability coupled with innate ROS responsiveness, these polymeric structures act as stimuli responsive materials, possessing a capacity to respond to local microenvironmental cellular cues. For example, polypropylene sulfide (PPS) possesses a thioether structure which exhibits an amphiphilic behavior once oxidized and has been leveraged in vesicles to achieve on demand drug delivery in the presence of ROS. Additionally, tellurium and selenium containing polymers have been produced in various microstructures to achieve similar goals as PPS. A particular structure which is of interest to us has been the thioketal (TK). TKs vary from “solubility-switch” PPS containing structures as the TK bond is cleaved upon oxidation, resulting in bulk degradation of TK containing structures. Poly(thioketal) (PTK) containing materials in biomedical research have been used in low molecular weight applications such as in injectable / moldable formulations, as bone cements through utilization of PTK urethane chemistries, in nanoparticles for anticancer drug delivery and oral delivery of therapeutics, polymer-dye conjugation, and more use cases which have been reviewed extensively.

[0055] While great efforts have been made to develop PTK based bone cements, some key issues persist with these formulations. PTK-urethane bone cements require a 3-step synthesis process, necessitating the use of toxic catalysts such as bismuth (III) chloride and lithium aluminum hydride. Much care needs to be taken when working with these precursors and even more diligence is required for the complete clearance of these catalysts for their use as tissue engineered constructs (TEC). The utilization of these catalysts leads to limited biocompatibility, rendering the technology unsuitable for clinical use.

[0056] Utilization of additive manufacturing (AM) techniques in biomedicine has resulted in the generation of TECs possessing complex biological structures and high spatial resolution for use in biomedicine. Various methods for achieving 3D printable bulk materials allow for the employment of a wide variety of chemistries possessing unique characteristics andproperties. One aspect of the present invention utilizes the photopolymerization 3D printing technique stereolithography (SLA). SLA necessitates the use of a UV curable resin, achieving complex morphology printing in a layer-by-layer manner through spatially specific UV light irradiation. Furthermore, thiol- ene click chemistry in 3D printing is a well-established field and is amenable to the limitations of PTK chemistries. At the lower end of the UV spectrum (254nm), thiolene polymerization occurs without the use of a photoinitiator. However, most resin printers do not possess such low wavelength UV sources due to high cost, therefore, printers usually possess UV sources with wavelengths from 365-405nm. Careful considerations need to be made as to what photoinitiator one is intending to implement in their resin mixture as there are photoinitiators specific to certain wavelengths. Photoinitiators catalyze thiolene photopolymerization reactions via a radical step-growth polymerization. Polymerization occurs when a thiyl radical created from an incident photon associates with a vinyl group substituting itself for the alkene functional group. The radical is then chain transferred to a free thiol which propagates the reaction. While great strides have been made to develop complex bioprinting techniques and materials, PTK based ROS degradable materials have yet to be translated to AM techniques.

[0057] In one embodiment, the present invention utilizes TK chemistries to address shortcomings in ROS degradable 3D printable polymers and recently developed PTK based bone cements. FIG. 1 illustrates an embodiment of the use of an inventive PTK in the present invention. Firstly, a PTK is synthesized in a one pot reaction through use of a minimally toxic and easily removable catalyst. Additionally, the inventive PTK monomer is incorporated into a bone cement material and SLA printable resin. PTK bone cements may utilize a commercially available tri-epoxy linker for combination with the CaP hydroxyapatite (HaP). Furthermore, thiol-ene photopolymerization chemistry can be utilized for SLA printing of ROS degradable TECs. In one embodiment, two commercially available alkene terminated monomers are combined with PTK, the photoinitiator Irgacure 819, and the radical scavenger TEMPO used as a photoabsorber to reliably print complex structures. The PTK containing TECs of the present invention demonstrate bio-responsive degradation using cell-produced ROS and achieve augmented healing outcomes within osseus tissue compared to the current clinical standards.ROS- responsive polymer systems

[0058] In one embodiment, the present invention has the goal of improving the functionality of ROS- responsive polymer systems. To this end, the molecular structure of ROS-degradablelinkers within polymer networks have been re-engineered to significantly enhance their applicability in drug delivery and 3D printing. The present invention involves the synthesis, validation, and initial in vivo testing of these engineered materials.

[0059] In one embodiment, the present invention relates thioketal bond presentation to oxidation-mediated degradation and drug release in responsive polymer systems. A library of TK linkers is synthesized from new precursors with enhanced hydrophilicity or therapeutic bioactivity and evaluated for sensitivity to ROS. These linkers are incorporated into cationic polymers to form polyelectrolyte multilayer drug coatings for ROS- triggered therapeutic delivery, which is quantified both in vitro and in vivo. The primary outcome of this work is the development and characterization of new TK-linked biomaterials that rapidly respond to physiologically-relevant levels of ROS for the controlled delivery of bioactive therapeutics.

[0060] In another embodiment, the present invention is used to create oxidation-degradable resins for 3D printing of responsive biomaterial implants. TK dithiol monomers are combined with branched -ene crosslinkers and a non-toxic photo-initiator to create resins that are compatible with stereolithography 3D printing. Print parameters are optimized for these resins to generate high-fidelity constructs that are non-toxic, easily fabricated, and selectively degraded by ROS. The primary outcome of this work is the creation of new photo-activatable resins that can generate oxidation-responsive biomaterial implants via 3D printing.Stimuli-Responsive Biomaterials in Regenerative Medicine

[0061] Synthetic polymers have been extensively used in regenerative medicine and are regularly formulated into erodible drug delivery systems or degradable scaffolds. In vivo degradation of these implants is most commonly facilitated by hydrolysis of ester bonds in the polymer structure and can also be simply modulated by tuning polymer crystallinity or hydrophilicity. However, these implants rely on pre-determined degradation rates encoded in the original formulation that may imperfectly translate into a heterogenous patient population. The resulting mismatch between rates of implant resorption and tissue regeneration can compromise overall healing, whether from prematurely degraded tissue engineering scaffolds or drug- loaded implants that prematurely released their therapeutic payloads. To this end, environmentally- responsive polymers have been extensively investigated as “smart” materials that react to specific biological stimuli, including enzymes, pH, or reactive oxygen species. Prominent examples include hydrogels degraded by specific cell-produced proteinases, gels with pH-responsive drug release to target inflamed tissues, and hydrogels with oxidation- triggered release of small molecule compounds.

[0062] Due to their selective production by cells and elevated presence in healing tissues, ROS such as hydrogen peroxide (H2O2), superoxide, or hypochlorite are attractive signals for facilitating specific biomaterial responsiveness. These highly reactive and short-lived molecules are important mediators in various biological processes and the immune response, and elevated ROS, or “oxidative stress”, is a hallmark characteristic of inflammation and pathogenesis in many diseases. Many synthetic polymers with oxidation responsiveness have been developed over the past two decades, including polysulfides, selenium-linked polymers, poly(oxalates), phenylboronic esters, oligoprolines, and thioketals. These materials have been successfully employed both as triggerable nano-scale drug delivery vehicles and bulk- scale biodegradable implants. However, their limited sensitivity and slow responsiveness to scarce biological signals remain a central bottleneck in their continued development. Creating reactive polymer systems that can quickly respond to biologically-relevant concentrations of triggering stimuli remains a critical goal in both fundamental and translational biomaterials research.Thioketal Linkers in Oxidation-Sensitive Polymer Systems

[0063] Since their introduction by the Murthy group in 2010, thioketal linkers have been incorporated into many polymeric biomaterials to confer ROS responsiveness. Conventional TK bonds are insensitive to hydrolysis but are selectively cleaved when exposed to ROS. This selective degradation mechanism has typically been used with triggerable nano-therapies to target high-ROS tissues such as intestinal lesions, inflamed muscles, or cancer cells. Efforts pioneered by the PI have also translated new poly(thioketal) (PTK) polymer formulations into bulk-scale biomaterials for multiple applications. These previously described TK polymer systems are all nearly inert in purely aqueous conditions but display dose-responsive activation when incubated with ROS. These include PTK-urethane scaffolds that facilitate cell-mediated material resorption and skin wound regeneration, PTK-crosslinked poly(ethylene glycol) (PEG) hydrogels for injectable stem cell delivery from a cell-degradable synthetic matrix, and conformal PTK coatings for on-demand delivery of therapeutic bone morphogenetic protein-2 (BMP -2) to bone defects. In short, employing simple yet versatile TK chemistry is a highly promising strategy for creating stimuli-responsive biomaterials.

[0064] However, TK polymers do feature significant hurdles that must be overcome for clinical translation. Most significantly, current TK-containing materials are insufficiently responsive to physiologically-relevant doses of ROS which are quantified at less than 0.1 mM in most tissues and feature in vivo lifetimes ranging from micro- to nano-seconds. Though all the PTK systems displayed robust ROS dose responsiveness in benchtop testing, only some of thenanoscale drug coatings achieved significant (though moderate) in vitro sensitivity at 0.1 mM H2O2. Consequently, all three of these formulations only manifested significant in vivo activity after weeks or months of implantation. Though prolonged material retention can be beneficial for scaffolds that promote soft tissue infiltration or hydrogels that protect encapsulated cells from immunotoxicity, many other biomaterial applications require significantly faster systems. Recent reports further highlight this need. The ROS-degradable drug coatings developed by the PI facilitated less bone growth than expected due to too-slow BMP -2 release kinetics in vivo, while PTK-based bone cements produced by Guelcher et al. featured negligible in vivo degradation and remodeling in slow-healing rat and rabbit bone defect models. In short, increasing the oxidative sensitivity of TK-based materials would represent a significant breakthrough for this technology.

[0065] Improving the responsiveness of ROS-responsive biomaterials through polymer engineering strategies has been explored in other systems, but essentially all TK-based technologies to date feature the same conventional linker structure. Crucial work from the Thayumanavan group recently elucidated the exact oxidative degradation mechanism behind TK bond cleavage, and also attempted to describe the relationship between bond structure and oxidative sensitivity. While this report provided numerous valuable findings, the lead- candidate TK structure from this work still maintained the same basic configuration and ROS responsiveness as the many previously-described TK biomaterials. However, the present invention has produced additional insight into their behavior, increasing material hydrophilicity (nanoscale coating > hydrogel > scaffold) also increases material oxidative responsiveness. Without being bound by theory, it is hypothesized that the inherently hydrophobic TK bond structure (featuring two non-polar pendant methyl groups) limits contact with ROS in aqueous environments both in vitro and in vivo. In short, increasing material hydrophilicity boosts ROS interactions with the degradable linker to improve stimuli responsiveness. This also extends to nano-scale PTK polymer systems, which possess extremely high surface area to volume ratios; this likely increases material interactions with water-borne ROS to consequently increase sensitivity to oxidation. While creating more hydrophilic polymer networks surrounding TK linkers remains a viable approach to increase ROS responsiveness, a more widely-translatable and effective strategy would be to re-engineer the degradable linker itself. The present invention introduces new configurations of oxidationsensitive bonds for the creation of materials that are highly responsive to biologically-relevant levels of ROS.3D Printing of Environmentally-Responsive Biomaterials

[0066] Over the last 30 years, additive manufacturing (i.e. 3D printing) has rapidly become established as a critical methodology for rapidly prototyping constructs with complex geometries. In the biomedical space, 3D printing has become popular in many applications but most notably for fabricating custom-fit tissue engineering scaffolds that can fill complex injury sites. As with many tissue engineering systems, 3D printed scaffolds are almost universally made from hydrolytically-degradable polymers. The most common examples include the polyesters poly(lactic-co-glycolic acid) (PLGA) and poly(caprolactone) (PCL) due to their demonstrated biocompatibility. As mentioned above, hydrolytically-degradable implants can suffer from a number of shortcomings including premature biodegradation before complete tissue regeneration is achieved. Despite this noted deficiency with water-sensitive polymers, cell-responsive materials that could better align scaffold resorption with biological activity have been relatively unexplored in 3D printing. This gap in technology development motivated the development of our inventive ROS-sensitive, TK-based 3D printing materials for application as cell-degradable tissue engineering scaffolds.

[0067] In designing TK materials that are compatible with 3D printing techniques, the two most common methodologies for polymer printing include fused deposition modeling (FDM, creates subsequent layers of extruded molten polymer) and stereolithography (SLA, photocrosslinking of a liquid resin to create subsequent layers of hardened material). Despite the explosion of PTK biomaterial systems developed over the past decade, only a single example of 3D-printable PTK scaffolds has recently been reported and requires complex synthetic workup alongside a custom 3D printing system. Since PTK polymers typically only achieve low molecular weight conversions and are often liquids at room temperature, they are unsuitable for extrusion-based FDM fabrication and are more compatible with SLA printing. Though the hardware and feedstock materials are slightly cheaper with FDM compared to SLA systems, SLA resins can often achieve finer resolution prints and offer a wider range of material properties. The present invention utilizes low molecular weight TK monomers with reactive dithiol end groups to create simple, low-cost, photo-activatable liquid resins that are compatible with commercial SLA 3D printing systems.Relating thioketal bond presentation to oxidation-mediated degradation and drug release in responsive polymer systems

[0068] Thioketal bonds have been incorporated into numerous biomaterial systems to confer selective ROS-responsiveness. However, the responsiveness of TK-linked polymers tophysiologic ROS concentrations (~0.1 mM) has been limited. Since more hydrophilic TK biomaterials are more sensitive to oxidative degradation, this work engineers different formulations of the TK bond itself. The engineered TK configurations will: 1) enhance sensitivity to oxidation by increasing linker hydrophilicity, and 2) directly release complexed bioactive compounds from the polymer backbone upon TK cleavage. These new materials are formulated into electrostatic coatings and evaluated for activity upon treatment with physiologic ROS doses both in vitro and in vivo.Synthesize and characterize a library of thioketal linkers with varying hydrophobicity

[0069] A new series of thioketal linkers are synthesized by a condensation reaction between amine-protected cysteamine monomers and ketone-containing molecules with various levels of hydrophilicity (Log P). These common ketones include acetone (base thioketal), levulinic acid (LEV-TK), pyruvic acid (PYR-TK), sulfonyl acetone (SA-TK), and oxoglutaric acid (OXO-TK).

[0070] Importantly, preliminary NMR experiments support the viability of the concept of relating linker hydrophobicity to oxidative degradation. When compared against the base TK linker (Log P = 2.61), more hydrophilic TKs made with levulinic acid (LEV-TK, Log P = 2.2) or pyruvic acid (PYR-TK, Log P = 2.15) experienced significantly faster degradation in ImM H2O2 but were completely inert without ROS treatment (data not shown). These results demonstrate the exciting potential of the present invention for improving TK material responsiveness.Fabricating the TK family

[0071] Based on the successful creation of base TK, LEV-TK, and PYR-TK linkers in preliminary investigations, the TK family is fabricated. To reiterate, the base TK configuration is the only currently used linker chemistry in TK-based responsive polymer systems. The TK formulations of the present invention, which are generated from different ketone precursors, are all completely novel materials. The generated TK linkers will display increased oxidative sensitivity with increased hydrophilicity while remaining inert to hydrolysis.Creating oxidation-degradable resins for 3D printing of responsive biomaterial implants

[0072] Biomaterial implants that undergo controlled biodegradation in vivo are foundational components of tissue engineering therapies and drug delivery systems. Recently developed cell-responsive polymer systems are highly promising in medical applications since they directly link material functionality with biological activity. Though advances in additivemanufacturing (i.e. 3D printing) have greatly expanded the geometries that can be achieved with certain polymeric biomaterials, to date there are very few literature examples of 3D- printable polymers that are environmentally-responsive. Here, we seek to employ our expertise in developing oxidation-sensitive polymer systems to create a family of ROS-degradable resins from thioketal precursors that are amenable to stereolithography (SLA) 3D printing. These resins will use photo-sensitive thiol-ene chemistry due to this reaction’s rapid progression, high efficiency, and uniform polymer network formation. These responsive resins will broadly impact both additive manufacturing and biomedical research by offering a new methodology for creating highly customized medical implants that dynamically interact with the local tissue environment.Synthesize and characterize a family of photo-curable resins made from TK precursors

[0073] To create oxidation-responsive resins that can be fabricated into complex biomaterial constructs, we first synthesize TK dithiol precursors that are compatible with -ene-terminated crosslinkers. Using the non- toxic dithiol monomer 3,6-dioxa 1,8 -octanedi thiol (DOT), low molecular weight TK dithiol linkers are synthesized using previously described methods. Two TK linkers are synthesized, the first featuring the standard acetone-based TK bond and the second featuring the lead-candidate TK bond with enhanced sensitivity as guided by previous results. These two linkers are characterized by NMR, GPC, and an Ellman’s assay to confirm linker structure, molecular weight, and thiol content. Three thiol- reactive crosslinkers featuring -ene chemistries (methacrylate and allyl ether) are combined with the TK dithiol linkers to assess these materials’ potential as resin formulations. Trimethylolpropane trimethacrylate and trimethylolpropane triallyl ether are commercially available at relatively low cost, feature the same core structure with three reactive groups for crosslinking, and have been used previously in 3D printing applications. The two TK linkers are respectively combined with the three separate -ene crosslinkers and the minimally-cytotoxic photo-initiator Irgacure 2959 and photo-inhibitor TEMPO to create the complete resin formulations (pilot TK- allyl ether resin shown in FIG. 6A).

[0074] Testing is used to evaluate resin viscosity values using parallel plate rheometry to ensure the liquid resins achieve less than 5 Pa- s viscosity, the estimated maximum that can be tolerated in SLA applications. To calculate optimal light irradiation parameters for 3D printing, cure depth of the resin formulations as a function of light exposure time is first determined using a photo-irradiator cure box with a 365nm light source and 5mW / cm2 power rating. Resins formulated with increasing doses of photo- initiator (0.1, 0.5, 1, 2 wt%) are added ontoa glass slide, irradiated for 5, 10, 15, or 20s, then washed in isopropyl alcohol to remove unreacted resin and determine the thickness of crosslinked material remaining. Since this photo-initiator generates free radicals, solidified samples are also be evaluated by solid state NMR to ensure that TK bonds have not been significantly damaged by light-generated radicals. Lead candidate formulations that quickly and reproducibly produce solidified samples and are not significantly degraded by radicals during photo-curing are carried forward for testing with SLA printing.Optimize print fidelity of new resins and characterize 3D printed constructs

[0075] With the establishment of viable TK-based resins, these materials are formulated into model constructs using SLA 3D printing. Our lab possesses a commercial SLA printer (AnyCubic Photon Mono 4K, 405nm LED light source, 4mW / cm2 power rating, 100cm2 print bed, 35pm nominal resolution) which is used to assess resin performance in model prints. Pilotscale prints generated from TK trimethacrylate resin are shown in FIG. 6B, confirming these materials are compatible with SLA printing. For initial testing, each resin is used to 3D print tugboat shapes as shown in FIG. 12A and FIG. 12B. The photo-absorber TEMPO is added to resins as needed to limit over-crosslinking and light scattering that can affect print resolution. Following best practice for biomedical SLA-printed materials, excess resin is removed from all final constructs with an isopropyl alcohol rinse before doing a post-print UV cure. Generated grid samples are imaged with optical and scanning electron microscopy (SEM) to assess strut diameter and pore size to quantify each resin’s print accuracy. Next, standard dog-bone samples for tensile testing are printed from the different resins to determine their mechanical properties, including elastic modulus, yield strength / strain, failure strength / strain, and toughness. To serve as hydrolytically-degradable control biomaterials, scaffolds with the same architecture are fabricated from commercial PLGA (75:25 L:G, lOOkDa) and PCL (50kDa) polymer filaments using our lab’s AnyCubic Kobra FDM printer. Both PLGA and PCL are high molecular weight thermoplastics that are most commonly fabricated with FDM systems in 3D printing applications.

[0076] With a small library of resins and the corresponding 3D printed materials established, in vitro testing for biodegradation and cytotoxicity is performed. Since all samples are formulated with ROS- sensitive TK precursors, printed grids are incubated in PBS with escalating doses of H2O2 (0, 10, 100, 1000 mM) to measure oxidation-mediated scaffold degradation in vitro. Sample degradation will also be qualitatively assessed by SEM imaging. Confirming these materials’ oxidative sensitivity, printed samples made from TK-trimethacrylate or TK-triallyl ether resins demonstrated robust ROS-mediated degradation in preliminary in vitro experiments (in FIG. 6C). Finally, printed scaffolds from the different resins are evaluated for cellular cytotoxicity. Following guidelines from ISO standard 10993- 5 (2009), scaffold samples are incubated in cell culture media for 24hrs before treating MC3T3- E1 cells with conditioned media to assess in vitro material toxicity. Analogous FDM-printed PLGA and PCL scaffolds will also be assessed for in vitro degradation and cytotoxicity to benchmark against the SLA-printed PTK materials.ROS degradable polymers

[0077] Conventional biodegradable materials have disadvantages such as degradation mediated by water and mis-matched wound area and material presence. ROS degradable materials, on the other hand, have cell-mediated biodegradation. They are an ideal form for material degradation matching wound size. The advantage of ROS degradable polymers compared to hydrolytically degradable polymers (water degradable) is a precise matching of wound area to the amount of scaffold remaining in the wound. With water degradable materials, an additional design consideration is needed which determines the degradation kinetics of the material implanted in a wound site. A material that degrades too fast leaves a wound untreated while a material that degrades too slowly does not clear the wound quick enough to allow for proper tissue reintegration. With ROS degradable scaffolds, this design consideration is not needed since material degradation is driven by cell produced molecules. Essentially this ties material degradation to wound tissue regeneration via cellular activity.ROS in the body

[0078] The present invention uses the strategy of leveraging the body’s innate healing mechanisms for bone. A molecule implicated in osteogenesis (bone regeneration) are reactive oxygen species (ROS), which is a cell signaling molecule. Thioketal (TK) is the ROS degradable component. When bulk material is created using TK containing precursor, the scaffolds structure degrades selectively to ROS molecules. This is unique compared to conventional hydrolytically degradable biomaterials. Combination of SCTK and the commercially available alkene terminated monomers: pentaerythritol allyl ether (AE, left most commercially available monomer) and trimethylolpropane trimethacrylate (TMA, center monomer) a liquid resin is created (goop) that fills the vat space above the 3D printer light source (see FIG. 7). In digital light printing (DLP), an LED array of UV light is used to create spatially specific patterns through the transparent bottom of the resin vat. The crosslinkingpolymerization happens and the final material structure is created in a layer by layer manner to achieve the final printed part.Resin fundamentals

[0079] Thiol-ene polymerization can be undertook by itself with UV <300nm. Most printers utilize 405nm or 365nm UV. FIG. 8 shows an embodiment of the composition of the resins that are useful in the present invention. Specifically, the photoinitiator irgacure 819 (BAPO) is incorporated which catalyzes the photocrosslinking reaction specifically at a 405nm wavelength. This was chosen because most commercial UV printers possess an array with 405nm light. There are commercially available printers with 365nm and other wavelengths however 405nm printers are the most common and the cheapest on the market. However, with just BAPO in the resin formulation, the crosslinking reaction does not stop and ends up crosslinking the whole resin vat. Therefore, a dampening component is used (called photoabsorber) which inhibits this reaction from continuing. In our application the photoabsorber we are using is TEMPO or specifically (2,2,6,6-Tetramethylpiperidin-l-yl)oxyl. Effort is made to find a combination of concentrations for these two components to achieve spatially specific prints that do not overcrosslink the resin.

[0080] FTIR Studies also demonstrated -ene terminated reagent stability in UV. No homopolymerization is observed (see FIG. 9). Referring to FIG. 10, FTIR was used to determine thiol elimination in UV and non-UV exposed samples. Ellmans was also used to confirm this result.

[0081] Further, we want our dithiol SCTK to only crosslink with the alkene terminated monomers instead of the alkene monomers reacting with themselves which is also a commonly used chemistry in the field of UV printing. FTIR studies were conducted to determine the structure of the resins before and after UV polymerization. Here we find that in the homopolymerization study done in FIG. 9, there is nor reduction of functional group peaks meaning there is no reaction happening between -ene terminated reagents under 405nm UV. However, in FIG. 10 we see reduction of -SH and -Ene peaks when incorporated together. This tells us the polymerization is happening how we believe it is, between thiol and ene groups. This is further corroborated in the degradation studies shown in the examples.EXAMPLESExample 1 : Development of a PTK bone cement for orthopedic implant fixation

[0082] To achieve appropriate mechanical properties of a PTK bone cement, the present invention improves the underlying PTK chemistry. It is well established that amorphous polymers containing higher crosslink densities demonstrate higher ultimate stress and lower ultimate strain. To utilize PTK containing structures in an osseus setting, PTK bone cements are optimized to achieve the highest possible elastic modulus to drive crosslink density as high as possible.

[0083] Briefly, PTK synthesis protocols involved an acid catalyzed reaction between 2,2 dimethoxypropane (DMP) and 2-mercapto ethyl ether (MEE) was carried out in a nitrogen environment heated to 79°C in an oil bath and allowed to stir for 16 hours. These protocols were favorable for assessing preliminary formulations of PTK bone cements, however, to achieve material properties analogous to bone tissue, modifications were required. To achieve the highest crosslinking density, PTKs molecular weight (Mw) must be driven towards its theoretical minimum which is 404.66Da. Previous synthesis protocols yielded Mw close to our minimum but within a range of 550-650Da. Elimination of heat and a reduction in reaction time to 5 hours were the main modifications to the synthesis. FIG. 2 shows a combined gel permeation chromatography (GPC) of varying protocols followed for synthesis of short-chain poly(thioketal) (“SC-PTK”). In the unoptimized curve, PTK elution time is earlier than others indicating a larger Mw and the area of the elution peak is greater demonstrating a larger poly dispersity index (PDI). This indicates a greater range of Mw and consequently introduces a large variability of final crosslinked materials. However, in optimized protocols, PDI is driven down along with Mw. Additionally, Mw was estimated utilizing a modified protocol of the Ellman’s assay. Ellman’s assay utilizes the chromogenic compound 5,5’-Dithio- bis-(2- nitrobenzoic acid) (DTNB) which reacts with free sulfhydryl’s in a slightly alkaline solution turning the solution yellow. Utilizing a thiolated monomer with known Mw to develop a calibration, thiol concentration can be measured, and a Mw estimated for other thiol containing monomers. Ellman’s assay was used to confirm a reduction of SC-PTK Mw following synthesis protocol changes and confirmed what was observed through GPC. After optimization of our one- pot SC-PTK synthesis, we translated our novel ROS-degradable monomer into a bone cement material for orthopedic implant fixation. To achieve this, the thiol-epoxy click reaction was exploited for selection of a crosslinking monomer. Thiol-epoxy click reactions are efficient, regioselective and widely applied in biomedical spaces as shape memory polymers, thin film crosslinking chemistries and bulk material production. The commerciallyavailable tri -functional epoxy containing monomer trimethylolpropane triglycidyl ether was selected to form our bulk polymeric material. A biocompatible base catalyst was needed to initiate the thiol-epoxy click reaction. Choline hydroxide has been used as a biocompatible base catalyst in previous studies and demonstrated mild reaction conditions and was amenable for our applications. Additionally, 50pm diameter HAp particles were selected to introduce osteoinductive and osteoconductive properties to our fixating material. Larger diameter 250pm HAp were initially used for fabricating cements, however, we observed that this particle diameter produced scaffolds with weaker mechanical properties compared to its smaller particle counterpart. To form these cements, PTK, TA, HAp, and choline are combined in a glass crystalizing dish and completely homogenized with a metal spatula. The loose cement paste is then transferred to a removable cylindrical mold and prepared for testing. FIG. 5 shows the final bulk scaffold that is received and shows the cement microstructure obtained through scanning electron microscopy (SEM). In FIG. 3 A, unoptimized TK and optimized TK sample moduli are compared for three different ceramic polymer mixes. Unoptimized PTK bone cements are significantly less stiff than their optimized counterparts. Human cortical bone possesses a compressive strength of 100-150MPa along the longitudinal axis while PMMA and CaP composite formulations demonstrate compressive strengths of 100-166 Mpa. In FIGs 3A and 3B, HAp and polymer content is modulated, and the properties assessed.

[0084] We determined a decrease in the ceramic component yields softer tougher scaffolds compared to higher ceramic containing formulations. This allows us to effectively modulate final mechanical properties through simple formulation changes thus expanding the versatility of our fixation material. Compared to native cortical bone, the compressive strength of our PTK bone cement is much less stiff, however, trabecular bone demonstrates an elastic modulus range of 3.5-125.6 MPa which is more amenable to PTK bone cement elastic modulus variability range given the data collected in FIGs 3A-C. Additionally, in polymer ceramic composite materials, it is understood that material properties increase over time due to extended curing times. Therefore, FIG. 3C analyzes cement properties as a function of time and demonstrates a plateau of properties with no significant differences after about 7-10 days of curing. FIG. 3D shows selective ROS degradation of our bone cement in H2O2 media and water. Cements in higher concentrations of H2O2 degrade quicker compared to lower H2O2 containing solutions. Additionally, cement mass is stable and does not degrade in non-ROS aqueous solutions.

[0085] When bone cement is utilized in the clinic during AJR surgeries and other procedures, it is imperative that fixation materials possess a known working time for clinicians to be ableto combine the precursor materials and apply it during an operation. Therefore, in FIG. 4A we have modulated concentrations of the catalyst choline to extend or shorten curing and working times. This was done using a tac free test where liquid and cement powder precursors are mixed, and their hardening deemed complete once the material does not stick to a metal spatula. Additionally, FIG. 4B assesses the lethal dose of choline hydroxide in conditioned media cocultured with MC3T3-E1 cells. The cell titer-glo assay assesses cell viability as a function of mitochondria activity and was used for all cytotoxicity experiments. An observed toxic effect of 50% is shown at about 7.5 mg / ml of choline hydroxide. This corresponds closely to about 14% by mass of choline. Our tac free time with 14% choline demonstrated a tac free time slightly greater than 15 minutes and this formulation was chosen as our lead candidate. Moreover, non-thermogenicity of PTK bone cements are shown in FIG. 4C where a slight increase in temperature is seen initially followed by a plateau and no temperature change greater than tac free times. Lastly, no cytotoxic effects are seen in PTK bone cement conditioned cell media following ISO 10993-5 on tests for in vitro cytotoxicity (FIG. 4D). In summary, with this data we have assessed the viability of PTK bone cement as an orthopedic implant fixation material. PTK bone cements possess lower moduli and greater toughness, ROS degradability, complete degradability, and non-thermogenicity compared to PMMA based counterparts.Example 2: Creation of a 3D printable SLA resin using TK monomers

[0086] We leveraged thiol-ene UV click chemistry due to its convenience for our specific application, its wide use in other 3D printing studies, and wide selection of commercially available alkene terminated precursors. A kinetic analysis of UV induced thiol-ene click chemistry was referenced to determine the appropriate -ene functionalized monomer for our intended use. Allyl ether and methacrylate terminated groups demonstrated acceptable reaction kinetics and these parameters were used for commercial reagent selection for photosensitive resin development. Trimethylolpropane triallyl ether (AE) and trimethylolpropane trimethacrylate (TMA) were selected and preliminary resin formulations were attempted. Additionally, our initial attempts incorporated the photo- initiator Irgacure 819 (BAPO). BAPO was used as a photoinitiator in other photo resin development studies and found to be minimally cytotoxic at 1% wt incorporation by mass. Furthermore, BAPO demonstrates peak absorbance at 405nm which is amenable to the Anycubic Mono 4K resin printer our lab uses. Concentrations of BAPO around 1% have yielded resins which over crosslink once exposed to a printing geometry.

[0087] Preliminary studies have roughly measured the mechanical properties of PTK-AE and PTK-TMA resins. Resins were deposited and cured on a glass microscope slide to form a 12x4xlmm resin sample. This was removed from the slide and loaded onto a mechanical tester to assess the material tensile properties as shown in FIG. 14E and FIG. 14F. PTK-AE films demonstrated higher toughness and yield strain possessing attributes similar to an elastomer, whereas PTK-TMA was more robust and failed at higher stresses. There was an artifact in the testing for PTK -TMA where a plateau of only strain and no stress is being measured. We believe this is due to improper loading and geometry of our sample. This test was done with the intention to assess the mechanical properties of the resins since there was a noticeable qualitative difference. We intend to fully characterize lead candidate resin formulations after further experimentation.

[0088] To assess resin printing parameters, cure depth as a function of UV energy can be measured for each formulation as shown in FIG. 14A-D. This provides insight into the innate parameters that should be used for printing such as layer depth, and exposure time for each layer. The following relation is established for determining cured depth: CD =Where CD is a measurement of the thickness of the sample, Dp is penetration depth, Ec is the critical energy exposure to induce crosslinking in the resin, and Eo is the exposure at the resin surface. Dp and Ec are used to inform UV exposure time and layer thickness when starting a print. These parameters were successfully measured using our printers UV source, timed exposure, and a micrometer to measure cured depth. Briefly, resins are deposited onto a glass microscope slide and placed on top of the 3D printers UV source. Microscope slides are imperative to use here due to their optical transparency and lack of light scattering. UV illumination time is attenuated to modulate UV energy deposited into the resin. The crosslinked samples adhere to the microscope slides while uncrosslinked resin is washed in acetone. The thickness of the polymerized zone is measured and the thickness of the slide is subtracted. These values are correlated with UV exposure time to determine cured depth and energy correlations. This data is plotted for preliminary formulations of our thiol-ene resins. Any changes made to the resin such as modulating photo initiator concentration and addition photo absorbers can change the curing properties. Therefore, cure depth is reevaluated once a lead candidate formulation has been chosen.Example 3 - The effects of PTK containing TECs within a critically sized rat calvarial model

[0089] The potential for the use of PTKs as a viable option for ROS degradable TECs has been established by data carried out through the development of PTK bone cements and photosensitive resins. A lead candidate for PTK bone cements has been identified, its negligible cytotoxicity measured, and properties assessed. A next step for PTK bone cements is to assess its bioactivity within a critically sized rat calvarial defect. An 8mm circular defect is created on rat skulls using a dental drill and trephine. Five rats per group are used with a total of 3 groups. One group receives a sham surgery where the skull portion is removed but not replaced with any material, a PTK bone cement group, and a PMMA HaP implant to compare between the inventive material and the clinical gold standard. Rats are housed for 10 weeks post operation and euthanized humanely for final material performance assessment. Histological analysis, and micro-CT scans are done to analyze tissue architecture after implant procedure and measurement of bone volume to compare tissue regeneration capacity between our groups. The PTK bone cements of the present invention should induce greater osteoinduction compared to PMMA counterparts and lead to preferred healing outcomes.Example 4: TK SLA Resins - Cured Depth

[0090] Cured depth experiments were conducted with a glass microscope slide and micrometer. We are not aware of any standard or widely accepted method for conducting cured depth experiments in the field of UV printing. This method was devised by the inventors and it was validated using another method which uses photo rheology. Referring to FIG. 12, a benchmark structure print was used to demonstrate the spatial resolution one can achieve with their printable resin.Example 5: Degradation of scaffold mass

[0091] Referring to FIG. 13, degradation data shows scaffold mass versus time of incubation. AT each time point, n=3 scaffolds are incubated in oxidative media which is meant to degrade the material over time. The higher the concentration of ROS in the media, the quicker the material will degrade. Dog bone samples (FIG. 13B) are printed to perform tensile tests and measure mechanical properties of each resin formulation.Example 6: Curing depth and UV dosage

[0092] Referring to FIG. 14, cured depth studies are done on the resin to determine the thickness of the material upon specific quantities of UV energy dosage. UV energy catalyzesthe resin reaction, therefore, with more UV energy a thicker sample is achieved. This data is used to determine the thickness of each layer when printing a more complex part on a UV printer. The illumination time determines the height of each layer in the Z axis, and the DLP LED array determines the spatial resolution in the X and Y axes.

[0093] The data presented herein shows that resins display a range of mechanical properties while all possessing ROS degradation capabilities. It has demonstrated an ability to print benchmark morphologies showing spatial resolution in (X, Y, Z).

[0094] Although not described in detail herein, other steps which are readily interpreted from or incorporated along with the disclosed embodiments shall be included as part of the invention. The embodiments that have been described herein provide specific examples to portray inventive elements, but will not necessarily cover all possible embodiments commonly known to those skilled in the art.

Claims

What is claimed is:

1. A short-chain poly (thioketal) (“PTK”) polymer produced by a method comprising: a. reacting 2,2 dimethoxypropane (DMP) or a ketone with a dithiol monomer in the presence of an acid using a nitrogen atmosphere, and b. stirring for a period of time from 1 to 72hrs, wherein the ketone is selected from the group consisting of acetone, levulinic acid, pyruvic acid, sulfonyl acetone, and oxoglutaric acid. The short-chain PTK polymer of claim 1 wherein the dithiol monomer is selected from the group consisting of 2-mercapto ethyl ether (MEE) and 3,6-dioxa 1,8 -octanedi thiol (DOT).3 A bone cement for orthopedic implant fixation comprising a composite of the short-chain PTK polymer of claim 1 and calcium phosphate (“CaP”). The bone cement of claim 3 wherein the composite comprises short-chain PTK polymer, a multifunctional epoxy linker, and hydroxyapatite (HAp) particles.5 The bone cement of claim 4 wherein the multi-functional epoxy linker comprises trimethylolpropane triglycidyl ether (“TA”).6 The bone cement of claim 4 wherein the composite further comprises choline hydroxide.7 The bone cement of claim 4 wherein the HAp particles have an average diameter of 50pm.8 A three-dimensional (“3D”) printable resin comprising a composite of the short-chain PTK polymer of claim 1 and a multi-functional linker possessing photopolymerization capabilities.9 The 3D printable resin of claim 8 wherein the multi-functional linker is selected from the group consisting of trimethylolpropane triallyl ether (AE) and trimethylolpropane trimethacrylate (TMA).10 The 3D printable resin of claim 8 wherein the composite further comprises Irgacure 819 (“BAPO”).11 The 3D printable resin of claim 8 wherein the composite further comprises 2, 2,6,6- tetramethylpiperidinyl-l-oxyl (“TEMPO”).12 A method of testing a 3D printed construct for treatment of a bone defect comprising implanting a 3D printed construct comprising the 3D printable resin of claim 8 into a rat calvarial bone defect and evaluating response factors comprising bone growth kinetics, implant degradation timelines, and local tissue response.

3. A method of testing a construct for treatment of a bone defect comprising implanting a construct comprising the bone cement of claim 3 into a rat calvarial bone defect and evaluating response factors comprising bone growth kinetics, implant degradation timelines, and local tissue response.

Images