Permeable barrier to enhance regeneration at tissue interfaces and therapeutic methods of use thereof

Abstract

Systems, methods, and compositions directed to the engineered incorporation of a tidemark-mimetic into a 3D printed osteochondral (OC)-mimetic hydrogel scaffold configured to recapitulate a mineralized barrier that restricts transport of calcifying ions into the articular (hyaline) cartilage layer, and / or an engineered cement line-mimetic configured to prevent cellular migration from the subchondral bone layer into the cartilage layers.

Description

[0001] PERMEABLE BARRIER TO ENHANCE REGENERATION AT TISSUE INTERFACES AND THERAPEUTIC METHODS OF USE THEREOF CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit of and priority to U.S. Provisional Application No.

[0003] 63 / 710,501, filed October 22, 2024. The entire specification and figures of the above-referenced application are hereby incorporated, in their entirety by reference.

[0004] STATEMENT OF FEDERALLY SPONSORED RESEARCH

[0005] This invention was made with government support under grant numbers R33HD090696 and 1R01AR069060 awarded by the National Institutes of Health. The government has certain rights in the invention.

[0006] TECHNICAL FIELD

[0007] The present disclosure is directed to the field of tissue growth and regeneration, and specifically a permeable barrier within a hydrogel scaffold configured to control cell and molecular transport at tissue interfaces.

[0008] BACKGROUND

[0009] Lesions to articular cartilage and underlying subchondral bone eventually result in osteoarthritis, a debilitating disease with no cure. A successful therapy will need to promote tissue regeneration, support integrative repair, and protect the surrounding tissue from further degeneration. As such, there is a long-felt need for a mechanically competent, stem cell-based regenerative approach to treat osteochondral (OC) defects. It has been previously shown in the art that OC-mimetic hydrogel can be designed that decouples the load bearing (i.e., structural) component from the soft cellular biomimetic component. This approach allows for a functionally graded, stiff structure with cartilage-matched mechanical stiffness, while independently the soft cellular niches are designed to direct mesenchymal stem cell (MSC) differentiation into the OC relevant tissues of cartilage and bone. Notably, OC-mimetic hydrogels induce rapid and targeted differentiation of exogeneous MSCs in vivo, enabling their direct participation in OC-tissue regeneration while simultaneously protecting and supporting integration with the surrounding tissue. Further, a cement line-mimetic can be engineered within the structural support of the hydrogel that is similar to the native cement line making it impervious to cell migration across the cartilage-bone interface, but pervious to nutrient transport. This structural configuration can protect the MSCs in the cartilage layer, enabling their rapid differentiation and contribution to regeneration.

[0010] SUMMARY OF THE INVENTION

[0011] In one aspect, the present disclosure describes systems, methods, and compositions directed to a permeable membrane that promotes tissue regeneration at tissue interfaces. In a preferred aspect, the disclosure describes the incorporation of a tidemark-mimetic into a 3D printed structure to recapitulate a mineralized barrier that restricts transport of calcifying ions into the articular (hyaline) cartilage layer. This tidemark-mimetic can be incorporated into an OC-mimetic hydrogel, for example, as described by Bryant et al., U.S. Patent No. 11298441 (incorporated herein by reference) with an engineered cement line a described herein, which can prevent cellular migration from the subchondral bone layer into the cartilage layers. By engineering both a cement line and a tidemark mimetic, the OC-mimetic hydrogel of the present disclosure can support three distinct tissue layers: (a) subchondral bone, (b) calcified cartilage, and (c) hyaline cartilage. Together, this novel class of OC-mimetic hydrogels can further improve the ability to achieve cell-mediated hyaline cartilage regeneration in the hydrogel.

[0012] BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1. Radiographs at 11 weeks post-injury revealed pronounced angular deformity and shorter limbs in the untreated group compared to the treated group (Fig. la). Limb length discrepancy in untreated limbs emerged at 5 weeks post-injury, while treated limbs showed differences from controls only at 7 weeks post-injury (Fig. lb). Treated rabbits generally had longer limbs, possibly due to age difference. Tibial angle decreased significantly in the untreated group from 2 weeks post-injury, but the treated group showed no significant difference until 7 weeks post-injury. *Treated vs. Control (Treated), *Untreated vs. Control (Untreated), # Treated vs. Untreated, p<0.05.

[0014] Figure 2A-B. Representative images showing different outcomes of composite implantation. The micro-CT and histological images are aligned to show the relative position of the implant. Alcian Blue-Hematoxylin (ABH) staining demonstrated fibrous repair tissue within the composite. While some rabbits had a displacement of the composite distal to the growth plate (Fig. 2a), in others, the composite remained in the growth plate area (Fig. 2b).

[0015] Figure 3A-F. Applicants used micro-CT to compare bone structural parameters between untreated and treated groups in full bone and growth plate (GP) defect areas. The figures demonstrate the differences between the right limbs (treated or untreated) and left limbs (controls). Most parameters showed no significant differences. However, in the GP defect area, trabecular thickness (Tb. Th) differed significantly between groups (p < 0.05), with the untreated group showing a greater increase compared to the treated group. This suggests the treatment specifically influences Tb. Th in the GP defect area.

[0016] Figure 4. Twelve male NZW Rabbits (6 wks. old) were subjected to a GPU in the right tibia (left=uninjured control). After three weeks, half of the animals underwent a second surgery to resect the bony bar that formed, followed by implantation of a 3D printed growth plate mimetic composite. The other animals were left untreated. Rabbits underwent weekly X-ray imaging (60kV, 0.4mA) and were euthanized 8 weeks posttreatment (or 11 weeks since GPI) to evaluate limb length and tibial angle. Micro-computed tomography at euthanasia assessed bone volume / tissue volume (BV / TV) within the injury area and within the composite. Repair tissue was evaluated with alcian blue hematoxylin (ABH) staining. Paired t-tests were used to compare injured limbs to the contralateral controls. Between treatment comparisons were evaluated with a linear mixed model. Significance was set at p < 0.05.

[0017] Figure 5A-D. (a) 3D-printed scaffold design, (b) Study groups with characteristics of 3D scaffold and CMH. (c) Tibia length and (d) tibial angle 2 weeks post-treatment. *vs. Control,# vs. Untreated, & vs. Group E, and+ vs. Group A, p<0.05.

[0018] Figure 6A-B. BV / TV within (a) the GPI area and (b) the composite 2 weeks posttreatment. ▲ vs. all groups, ■ vs. Groups C, D, E, p<0.05.

[0019] Figure 7. ABH staining of tibia 2 weeks post-treatment.

[0020] DETAILED DESCRIPTION OF THE INVENTION

[0021] Tissue interfaces, which are present throughout the body, connect two distinct tissues that have different biochemical and mechanical properties. Examples include bone-to-cartilage in articulating joints and the growth plate, bone-to-tendon, bone-to-ligament, and skin-to-muscle. When engineering multi-type tissues, creating distinct regions or layers of tissues can be challenging. While gradient scaffolds have been designed to capture key biochemical and mechanical properties, cells can migrate, and molecules can diffuse within a scaffold leading to ill-defined regions at interfaces.

[0022] To address this shortcoming, the present disclosure provides for a polymeric physical barrier that is designed independent of the tissue engineering scaffold (that is designed for cells to promote newtissue growth). The permeable barrier can be designed to prevent cell migration while allowing for tight control over molecular diffusion to permit selective transport of molecules based on size and / or charge. More specifically, in a preferred embodiment the present disclosure describes systems, methods, and compositions directed to a permeable barrier within an osteochondral (OC)-mimetic hydrogel scaffold configured to prevent cell migration while simultaneously controlling selective transport of molecules involved in cell signaling is novel. The barrier of the disclosures can be fabricated using grayscale patterning coupled with resin chemistry and / or extent of degradation.

[0023] As described herein, a permeable barrier can be fabricated using the 3D printing method of digital light projection, which uses patterned light to print a three-dimensional scaffold with controlled architecture. Using grayscale patterning and a hydrophobic resin, regions with high conversion served as the structural component, while regions of low conversion served as the permeable barrier. At high conversion, a highly crosslinked polymer is present, which restricts cell migration across the interface due to the tight polymer mesh while its hydrophobicity limits molecular transport. On the contrary, the low conversion regions produce a lower crosslinked polymer whose mesh size can be controlled by the degree of conversion. This allows selective transport of molecules based on size, but still prevents cell migration. In additional embodiments, the chemistry of the resin composition can be modulated, such as through the use of negatively or positively charged polymers to control transport of molecules across the barrier by charge in addition to size.

[0024] In another embodiment, degradable resin chemistries, such as through poly(P-amino esters) (PB AE)-acrylates, can be used. In this embodiment, (PBAE)-acrylates resins partially degrade and undergo a transition from a largely neutral chemistry to a negatively charged chemistry. In this embodiment, by spatially patterning the PBAE-acrylate resins, regions of low conversion can be more susceptible to degradation, while regions of high conversion can be more protected from degradation. When exposed to accelerated degradation conditions for short periods of time, the low conversion regions partially degrade and produce negatively charged functional groups. Using this approach, applicants demonstrate the ability to create local regions of negatively charged polymer without affecting the chemistry of the high crosslinked, structural regions. These methods can allow for 3D printing of a permeable barrier within a 3D printed scaffold to allow for selective transport while preventing cell migration across the barrier. The barrier can be 3D printed at any point in the scaffold depending on the tissue interface of interest.

[0025] As used herein, each of the following terms has the meaning associated with it in this section.

[0026] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, exemplary methods and materials are described.

[0027] Generally, the nomenclature used herein and the laboratory procedures in pharmacology and tissue engineering are those well-known and commonly employed in the art.

[0028] As used herein, the articles “a” and “an” refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. By way of example, “an element” means one element or more than one element.

[0029] As used herein, the term “about” is understood by persons of ordinary skill in the art and varies to some extent on the context in which it is used. As used herein when referring to a measurable value such as an amount, a temporal duration, and the like, the term “about” is meant to encompass variations of ±20% or ±10%, more preferably ±5%, even more preferably ±1%, and still more preferably ±0.1% from the specified value, as such variations are appropriate to perform the disclosed methods.

[0030] As used herein, the term “bony bar” or “physeal bar” is a premature physeal arrest, often resulting from injury or infection to an unfused physis. The bony bar consists of a bony bridge that crosses the growth plate and can result in growth abnormalities and deformities.

[0031] As used herein, the term “composition” or “pharmaceutical composition” refers to a mixture of at least one compound useful within the invention with a pharmaceutically acceptable carrier. The pharmaceutical composition facilitates administration of the compound to a patient or subject. Multiple techniques of administering a compound exist in the art including, but not limited to, intravenous, oral, aerosol, parenteral, ophthalmic, nasal, pulmonary, and topical administration.

[0032] As used herein “crosslinking” is meant to be a process of creating a bond that links one polymer chain to another. As described herein, a crosslinking agent can accomplish crosslinking. As used herein “crosslinking agent” or “crosslinking source” is meant to be an agent that is capable of forming a chemical or ionic links between molecules. Nonlimiting examples of crosslinking agents or sources include calcium chloride; ammonium persulfate (APS) and tetramethylethylenediamine (TEMED), glutaraldehyde, epoxides, oxidized dextran, p-azido benzoylhydrazide, N[a-maleimidoacetoxy] succinimide ester, p-azidophenyl glyoxal monohydrate, bis-[P-(4-azidosalicylamido)ethyl]disulfide, bis[sulfosuccinimidyl]suberate, dithiobis[succinimidyl proprionate, disuccinimidyl suberate, l-ethyl-3-[3-dimethylaminopropyl] carbodiimide hydrochloride (EDC), N-hydroxysuccinimide (NHS), visible light irradiation, ultraviolet irradiation, and combinations thereof.

[0033] A “disease” as used herein is a state of health of an animal wherein the animal cannot maintain homeostasis, and wherein if the disease is not ameliorated then the animal's health continues to deteriorate.

[0034] A “disorder” as used herein in an animal is a state of health in which the animal is able to maintain homeostasis, but in which the animal's state of health is less favorable than it would be in the absence of the disorder. Left untreated, a disorder does not necessarily cause a further decrease in the animal's state of health.

[0035] As used herein, the term “gel” refers to a three-dimensional polymeric structure that itself is insoluble in a particular liquid, but which is capable of absorbing and retaining large quantities of the liquid to form a stable, often soft and pliable, but always to one degree or another shape-retentive, structure. When the liquid is water, the gel is referred to as a hydrogel. Unless expressly stated otherwise, the term “gel” will be used throughout this application to refer both to polymeric structures that have absorbed a liquid other than water and to polymeric structures that have absorbed water, it being readily apparent to those skilled in the art from the context whether the polymeric structure is simply a “gel” or a “hydrogel.”

[0036] As used herein, the term “growth plate” refers to the epiphyseal plate or the hyaline cartilage plate in the metaphysis at each end of a long bone. The growth plate is the portion of the bone where new bone growth takes place, thereby elongating the bone. The terms “growth plate” and “physis” are to be used interchangeably. As used herein, the term “growth plate injury” refers to an injury to the epiphyseal plate or the hyaline cartilage plate in the metaphysis at each end of a long bone. The terms “growth plate injury” and “physeal injury” are to be used interchangeably.

[0037] The terms “patient,” “subject” or “individual” are used interchangeably herein, and refer to any animal, or cells thereof whether in vitro or in situ, amenable to the methods described herein. In a non-limiting embodiment, the patient, subject or individual is a human.

[0038] As used herein, the term “pharmaceutically acceptable” refers to a material, such as a carrier or diluent, which does not abrogate the biological activity or properties of the compound, and is relatively non-toxic, i.e., the material may be administered to an individual without causing undesirable biological effects or interacting in a deleterious manner with any of the components of the composition in which it is contained.

[0039] The term “prevent,” “preventing” or “prevention,” as used herein, means avoiding or delaying the onset of symptoms associated with a disease or condition in a subject that has not developed such symptoms at the time the administering of an agent or compound commences.

[0040] A “therapeutic” treatment is a treatment administered to a subject who exhibits signs of pathology, for the purpose of diminishing or eliminating those signs.

[0041] As used herein, the term “treatment” or “treating” is defined as the application or administration of a therapeutic agent, i.e., a compound of the invention (alone or in combination with another pharmaceutical agent), to a patient, or application or administration of a therapeutic agent to an isolated tissue or cell line from a patient (e.g., for diagnosis or ex vivo applications), who has a condition contemplated herein, a symptom of a condition contemplated herein or the potential to develop a condition contemplated herein, with the purpose to cure, heal, alleviate, relieve, alter, remedy, ameliorate, improve or affect a condition contemplated herein, the symptoms of a condition contemplated herein or the potential to develop a condition contemplated herein. Such treatments may be specifically tailored or modified, based on knowledge obtained from the field of pharmacogenomics.

[0042] As used herein, the term “therapeutically effective amount” refers to an amount that is sufficient or effective to prevent or treat (delay or prevent the onset of, prevent the progression of, inhibit, decrease or reverse) a disease or condition described or contemplated herein, including alleviating symptoms of such disease or condition. As used herein, the term “engineered” refers to a non-naturally occurring or man-made composition or component.

[0043] Throughout this disclosure, various aspects of the invention may be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention. Accordingly, the description of a range should be considered to have specifically disclosed all the possible sub-ranges as well as individual numerical values within that range and, when appropriate, partial integers of the numerical values within ranges. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed sub-ranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 2.7, 3, 4, 5, 5.3, and 6. This applies regardless of the breadth of the range.

[0044] EXAMPLES

[0045] Example 1: 3D-Printed Growth Plate Mimetic Composite Mitigates Early Growth Deformities.

[0046] Growth plate injuries (GPI) present significant challenges in pediatric orthopedics, as they can result in bony bar formation with subsequent growth arrest and limb deformities. Current treatment modalities are limited in their efficacy and fail to regenerate the complex structure of growth plate cartilage, highlighting an urgent need for innovative regenerative approaches. Applicant have developed a 3D-printcd grow th plate mimetic composite that improves limb lengthening and reduces angular deformity after GPI . The composite contains a 3D-printed pillar design structure that provides mechanical support and is infilled with a soft degradable cartilage -mimetic hydrogel (CMH) that provides chondrogenic cues. To prevent cell infiltration, Applicants demonstrated the 3D-printed structure design having has a top and bottom layer forms a physical boundary, allowing nutrient transport, but no migration of cells. The composite was tested in a rabbit model of growth plate injury and outcomes such as limb length, angular deformity, and repair tissue characterization were assessed.

[0047] The composite was fabricated using the following steps and materials: A modified triply periodic minimal surface (TPMS) gyroid-sheet structure with 1.25 mm unit cell size and 25% relative density 'as designed for the structure and sandwiched between two 50 pm thin layers that form a physical boundary. A photopolymerizable resin consisting of poly(P-amino ester) diacrylate (PBAE-dA) macromer, 0.85 wt% diphenyl-(2,4,6-trimethylbenzoyl)phosphine oxide (TPO) as the photoinitiator, and 0.15 wt% Quinoline Yellow (QY) as tire UV absorber was used with a custom projection-based microstereolithography 3D printer. The printed structure was infilled with a soft degradable CMH consisting of 18 wt% 8-arm, 10 kDa polyethylene glycol)-norbomene with tethered TGFP3, GCRGDS, chondroitin sulfate and MMP sensitive crosslinker.

[0048] As shown in Figure 4, twelve male NZW Rabbits (6 wks. old) were subjected to a GPI in the right tibia (left-uninjured control). After three weeks, half of the animals underwent a second surgery to resect the bony bar that formed, followed by implantation of a 3D printed growth plate mimetic composite. The other animals were left untreated. Rabbits underwent weekly X-ray imaging (60kV, 0.4mA) and were euthanized 8 weeks post-treatment (or 11 weeks since GPI) to evaluate limb length and tibial angle. Microcomputed tomography at euthanasia assessed bone volume / tissue volume (BV / TV) within the injury area and within tire composite. Repair tissue was evaluated with alcian blue hematoxylin (ABH) staining. Paired t-tests were used to compare injured limbs to the contralateral controls. Between treatment comparisons were evaluated with a linear mixed model. Significance was set at p < 0.05.

[0049] Radiographs taken at the end of the study (11 weeks post-injury or 8 weeks post-treatment) showed pronounced angular deformity and a shorter limb in the untreated group compared to the treated group (Fig. la). Limb length discrepancy between the untreated limbs and their contralateral uninjured controls emerged at 5 weeks post-injury and continued until the end of the study (Fig. lb, blue lines), while the treated limbs were significantly different from their controls starting only at 7 weeks post-injury (4 weeks post-treatment) (Fig. lb, orange lines). At almost all time points, treated rabbits had longer limbs than untreated rabbits, possibly due to being 5-7 days older. Tibial angle decreased significantly in the untreated group compared to its control as early as 2 weeks post-injury, while the treated group did not have a significant difference from its control until 7 weeks post-injury (4 weeks post-treatment). Bone tissue within the injured growth plate area was observed in both groups, but microCT did not detect bone tissue within the composite in the treated group (Fig. 2, purple outline). ABH staining demonstrated fibrous repair tissue within the composite, and no significant cartilage tissue. While some rabbits had displacement of the composite distal to the growth plate (Fig. 2, top row), in others the composite remained in the growth plate area (Fig. 2, bottom row).

[0050] Tire above data demonstrate that Applicants 3D-printed biomimetic composite can mitigate skeletal deformities seen after GPI in a rabbit model, by delaying the onset of limb length discrepancy and tibia angulation compared to an untreated group. Maintaining the composite within the growth plate area can prolong its ability to prevent skeletal deformities long-term, and the lack of bone formation within the composite further indicates its ability to prevent bony bar formation and restoring normal bone growth.

[0051] Example 2: Optimization of a 3D-Printed Growth Plate Mimetic Composite for the Treatment of Growth Plate Injuries.

[0052] The growth plate (or physis) is a cartilage layer near the ends of long bones in children and responsible for longitudinal bone grow th. Growth plate injuries (GPI) can result in bony bar formation and subsequent skeletal deformities. Applicants have developed a 3D-printed growth plate mimetic composite that improves limb lengthening and reduces angular deformity after GPI. The composite consists of a 3D-printed pillar design scaffold that provides mechanical support and is then infilled with a soft degradable cartilage-mimetic hydrogel (CMH) that provides chondrogenic cues. Applicants designed the scaffold based on a modified triply periodic minimal surface (TPMS) gyroid-sheet structure, which can resist shear loading better than pillar designs known in the art and decrease bone formation within the composite. Applicant’s composite incorporated three modifications: (1) incorporation of a physical boundary layer at the top and bottom of the scaffold to prevent cell ingrowth; (2) increasing the crosslinking density of the CMH to slow cell ingrowth, and (3) pre-polymerizing the CMH within the scaffold ex vivo instead of in situ to ensure complete polymerization. Hrese different composites were tested in a rabbit model of growth plate injury and outcomes such as limb length, angular deformity, and repair tissue characterization were assessed.

[0053] A modified TPMS gyroid-sheet structure with 1.25 mm unit cell size and 25% relative density was designed for the scaffold and sandwiched betw een two 50 pm thin layers with 0.9 mm x 0.4 mm rectangular cutouts (nTop) (Fig. 5a). Cutouts w ere aligned with the micropores of the TPMS geometry to enable infilling with low -conversion material to create a physical boundary layer that allows nutrient transport, but not migration of cells. For scaffolds without the boundary layer, the cutouts were left empty resulting in open space for cells to migrate. A photopolymerizable resin consisting of poly(P-amino ester) diacrylate (PBAE-dA) macromer, 0.85 wt% diphenyl-(2,4,6-trimethylbenzoyl)phosphine oxide as the photoinitiator, and 0.15 wt% Quinoline Yellow7as the UV absorber was used with a custom projection-based microstereolithography 3D printer. The scaffold w as infilled w ith a soft degradable CMH consisting of 8-arm, 10 kDa poly(ethylene glycol) -norbomene at a 10 wt% (normal crosslinking) or 18 wt% (high crosslinking) with tethered TGFP3, GCRGDS, chondroitin sulfate and MMP sensitive crosslinker.

[0054] Thirty-six male NZW Rabbits (6 wks old) received a GPI in the right tibia (left-uninjured control) and either left untreated or immediately treated with one of five composites outlined in Fig. 5b; Groups A-E (n=5-7 / grp). Composites differed in whether they had a physical boundary layer, which CMH was used (normal or high crosslinking), and whether the CMH was polymerized ex vivo or in situ. Rabbits were euthanized 2 weeks post-treatment. X-ray imaging evaluated limb length and tibial angle. Micro-computed tomography assessed bone volume / tissue volume (BV / TV) within the injury area and within the composite. Repair tissue was evaluated with alcian blue hematoxylin (ABH) staining. Paired Student’s t-test with control limbs and mixed model analysis with post-hoc Tukey’s HSD determined differences between groups (p<0.05).

[0055] Group A had shorter limb lengths than its uninjured control and Group E, while Group B had shorter limbs than untreated and Group E. Interestingly, Group E had longer limbs than its uninjured control (Fig. 5c). As a result of GPL the untreated group appeared to have more pronounced angular deformity compared to the control, but it was not significant (Fig. 5d). Implantation of a 3D-printed biomimetic composite appeared to mitigate angular deformity, with all groups, except for Group B which had a trend of decreased tibial angle compared to control (Fig. 5d). Group E had significantly more bone than other groups within the GPI area (Fig. 6a). When measuring only the bone in the composite, Groups A and B had significantly greater bone than Groups C, D, and E (Fig. 6b). Histological analysis revealed bone infiltration in composites of Group A, with some bone in Group B. Sparse areas of cartilage-like tissue within the composite were observed in Groups B-E, but no group exhibited significant growth plate cartilage regeneration in the GPI area (Fig. 7).

[0056] As described below, Applicants can further modify the 3D-printed growth plate mimetic composite for GPI treatment by incorporating a physical boundary layer, increased CMH crosslinking, and prepolymerization before implantation to prevent bony repair tissue formation and subsequent skeletal deformities. Composites with a boundary layer and / or CMH with high crosslinking and polymerization ex vivo (Groups C, D, and E) exhibited better preservation of limb length and tibial angles after GPI. While no group demonstrated significant growth plate cartilage regeneration w ithin the 2-week follow-up, Groups C, D, and E showed reduced bone tissue infiltration into the composite, indicating enhanced potential for preventing bony bar formation.

[0057] Example 3 : Experimental Rationale and Overview for engineered permeable membrane having a tidemarkmimetic.

[0058] The osteochondral unit is a complex biological system that connects articular cartilage to the underlying subchondral bone via a calcified cartilage layer that is approximately 20-250 pm thick in humans. Uris layer is demarcated by an upper and lower boundary of highly mineralized tissue called tire tidemark and the cement line, respectively. The tidemark separates calcified and non-calcified layers of articular cartilage in the OC unit and is about 5- 10pm thick. The tidemark is permeable to small solutes, but its anionic character restricts transport of cationic solutes. The tidemark is thought to serve an important function in the OC unit by preventing mineralization, likely due to the restricted transport of calcifying ions, and blocking invasion of blood vessels into hyaline cartilage. As described herein, Applicants have engineered an artificial tidemark into an OC-mimetic hydrogel that recapitulates the mineralized barrier of the native OC unit that reduces transport of calcify ing ions and enhances hyaline cartilage regeneration. In a preferred embodiment, a tidemark structure is introduced into a 3D printed structural support, which through its chemistry leads to ex vivo mineralization that recapitulates the native tidemark. As such, the ability to recreate a tidemark in in OC tissue via an engineering approach allows the presently disclosed methods and compositions to enhance hyaline cartilage regeneration in vivo. The mineralization capacity and corresponding transport properties in the tidemark-mimetic can be assessed as a function of the 3D printing resin chemistry and post-processing. The ability of the mineralized barrier to support hyaline chondrogenesis by MSCs can further be determined in vitro. Tire combined tidemark and cement link-mimetics in the full OC-mimetic hydrogel can be tested in tire rat OC defect model.

[0059] Example 4: Mineralization and molecular transport as a function of polylB-amino ester) chemistry'.

[0060] Applicants describe the use of a partially degraded poly(P-amino ester) (PBAE) network to form a 3D printed structure having a negatively charged polymer network that initiates mineralization ex vivo and decreases molecular transport of cationic molecules. In this embodiment, bulk prepared polymers from tire PBAE-diacrylate resin can be formed with varying initial crosslink densities, partially degraded, and tested for mineralization capacity. Once mineralized, molecular transport can be assessed as a function of molecule size and charge. The link between initial material properties (i.e., crosslink density), degradation conditions (pH and time), and mineralization capacity can be further determined. This study will determine which properties and conditions lead to tire formation of the tidemark-mimetic without negatively impacting the overall 3D printed structural mechanics and inducing mineralization in unwanted regions (notably tire cartilage layer).

[0061] Example 5: degradation and swelling properties of bulk prepared PBAE materials as a function of initial crosslink density'. pH, and time.

[0062] Crosslinked PBAEs degrade when an ester bond in the crosslinks is cleaved by hydrolysis, producing a hydroxyl and a carboxyl group. Since mineralization relies on negatively charged molecules, previous studies have shown that hydroxyl and carboxyl groups can act as nucleation sites for mineralization. PBAEs become increasingly negatively charged as degradation proceeds. Notably, by spatially altering the initial crosslink density, it is possible to control when and where the transition to a negatively charged polymer network occurs. Thus, by controlling both the initial crosslink density and the extent of degradation, it is possible to locally alter the chemistry' of the PBAE to induce mineralization in select regions. To this end, the initial crosslink density can be varied during photopolymerization by controlling the extent of conversion (via light intensity) of the PBAE-diacr late monomer into a polymer. Accelerated degradation conditions can be used to induce and control degradation through pH of the buffer and time. The hydrophobic (B6) PBAE-diacrylate monomer formed from the Michael-Aza reaction of butanediol diacrylate and benzhydrazide, which is used to prepare the 3D printed structure, can be implemented for tire same.

[0063] Bulk prepared B6 PBAE crosslinked polymers (5 mm diameter x 1 mm thick disks) can be formed at varying degrees of conversion (e.g., 10, 15, 20, 30, 50, 70, 100%) as measured by Fourier-transform infrared spectroscopy (FTIR). Each material can further be subjected to accelerated degradation conditions at increasing pH (8-11). At select time points, disks can be rinsed and reswollen to measure the compressive modulus in physiological buffer (pH 7) and the volumetric swelling ratio 0 in physiological buffer (pH 7, Ob) and in deionized water (pH 7, Qw). Tire ratio Q Qb can initially be near unity indicating minimal difference in swelling in buffer or water, as the B6 polymer is hydrophobic and neutral. At a critical time tc, Q Qb begins to increase due to the formation of fixed negative charges that lead to charge-charge repulsion of the network in water (the charges are shielded in buffer). ( „ / (?b and tccan be determined as a function of initial crosslink density, degradation pH, and degradation time. This allows identification of the initial crosslink densities and degradation conditions that lead to a highly negatively charged polymer, but not for 100% conversion under the same degradation conditions (i.e., tire bulk of the 3D printed structure is not negatively impacted). Under these conditions, the mechanical properties of the 3D printed structure are retained, while altering the chemistry locally in the tidemark region.

[0064] Example 6: Assess mineralization capacity of partially degraded PBAE materials.

[0065] In vitro mineralization can occur using simulated body fluid (SBF). Specifically, SBF is rich in ions (i.e., sodium, potassium, magnesium, calcium, chlorine, bicarbonate, and hydrogen phosphate ions) and in the presence of a nucleating site can lead to mineralization. Mineralization occurs in three stages: (a) accumulation of calcium cations adsorbed to anionic groups, (b) precipitation of Ca-P, and (c) crystal grow th. Immersion time in SBF influences the Ca / P stochiometric ratio in the cry stal and detennines extent of hydroxyapatite formation. A subset of initial crosslink densities of the B6 PBAE can be selected from the structures described above and subjected to accelerated degradation conditions and at select times, can be tested for mineralization capacity by placing the disks in SBF for up to three weeks. Weekly, samples can be removed and quantified for total Ca and P deposits. Raman spectroscopy can be used to determine the type of Ca / P crystal formed. In sum, the aforementioned steps can determine the extent of degradation required to induce mineralization.

[0066] Example 7: Assessment of molecular transport in the mineralized tidemark-mimetic.

[0067] Molecular transport can be studied in a subset of the structures described above via onedimensional diffusion testing system described by Bryant et al., (15-16) Briefly, PBAE-dA can be polymerized in the apparatus described by Bryant, degraded for a prescribed period of time, and exposed to simulated body fluid to create the mineralized tidemark barrier. Transport of different fluorescently labeled neutral dextran molecules ranging in molecular weight (e.g.. 3k-70k) and charged small molecules (e.g., negatively charged calcein, positively charged rhodamine) can be injected on one side of the device. Diffusion of the injected molecules can be monitored as a function of time by fluorescence (e.g., Versadoc), and can be calculated for each molecule by fitting the data (concentration vs time) to Fick’s second law of diffusion for one -dimensional in cartesian coordinates. In this embodiment, the transport properties across the tidemark-mimetic as a function of the degree of mineralization can be determined. Example 8: Effect of the tidemark -mimetic on chondrogenesis of MSCs in the OC-mimetic hydrogel. A dense mineralized barrier formed by the tidemark-mimetic reduces the transport of calcium and phosphate ions across the interface and protects MSC chondrogenesis from hypertrophy in the OC-mimetic hydrogel. Presence of high levels of calcium and phosphate ions in articular cartilage can induce mineralization and lead to hypertrophy. As noted previously in the art, small ions can readily diffuse across the bone-cartilage interface in the 3D printed structure of the OC-mimetic hydrogel and could affect chondrogenesis. As such, the use of a mineralized barrier through the tidemark can reduce transport of calcium and phosphate ions across the interface and decrease the propensity for MSC hypertrophy.

[0068] In this embodiment, MSC chondrogenesis can be assessed as a function of the mineralized tidemark-mimetic. A bilayer 3D printed structure containing tire tidemark (with no cement line) can be fabricated, partially degraded, and immersed in simulated body fluid for three weeks. Tire lattice of the tidemark can be formed from partially (e.g., 30%) converted PBAE and immersed in a basic solution (e.g., pH 8) for up to 2 days. The mineralized tidemark can be confirmed by scanning electron microscopy. The extent of transport of calcium and phosphate ions across tire interface can be first characterized by placing a high concentration of each ion separately and then combined in the lower chamber. Tire concentration of ions can be assessed in the upper chamber as a function of time using standard spectroscopy methods. To test the ability of the mineralized tidemark-mimetic to protect MSC chondrogenesis from hypertrophy can be tested in four experimental groups: (a) open lattice, (b) lattice with 30% conversion, but no degradation, (c) 30% conversion with degradation, and (d) 30% conversion with degradation and immersed in SBF. The first three experimental groups can be permissible to small ion transport, while the latter with the mineralized barrier can restrict transport. Human MSCs can be encapsulated in the cartilage-mimetic hydrogel in the top layer of the 3D printed structure. Chondrogenesis can be assessed for hyaline articular cartilage (Sox9, Aggrecan, Collagen II) and calcified cartilage (Runx2, Vegf, MMP13, Collagen X) by qPCR, biochemical assays, and (immuno (histochemistry.

[0069] In this configuration, when the lattice structure is open, partially polymerized, or degraded, the MSCs in the cartilage-mimetic hydrogel will undergo chondrogenesis but transition to hypertrophy, which will correspond to elevated diffusion of calcium and phosphate ions across the interface. On the contrary, the mineralization barrier can restrict transport of calcium and phosphate ions and prevent hypertrophy enabling the MSCs to undergo chondrogenesis and retain a hyaline cartilage phenotype.

[0070] Example 9: Assessment of the combined cement line-mimetic and tidemark-mimetic in the OC-mimetic hydrogel on cartilage regeneration in vivo.

[0071] The presence of a mineralized tidemark-mimetic enhances chondrogenesis and hyaline cartilage regeneration while supporting calcified cartilage regeneration in the region in between tire cement line and tidemark mimetics. Very few studies have incorporated a tidemark design into a scaffold design for cartilage or OC tissue engineering, but it has been suggested that the tidemark is critically important to the normal function of cartilage. As a result, an artificial mineralized barrier engineered into the OC-mimetic hydrogel, which mimics the native tidemark, can be used to improve cartilage regeneration in vivo.

[0072] In this preferred embodiment, a 3D printed structure containing a tidemark and cement line can be fabricated, partially degraded, and immersed in simulated body fluid for three weeks. Tire lattice of the tidemark can be formed from partially (e.g.. 30%) converted PBAE-dA, while the cement line can be formed from partially (e.g., 75%) converted PBAE-dA. The structure will be immersed in a basic solution (e.g., pH 8) for up to 2 days. Mineralization at the tidemark interface can be confirmed by SEM. An acellular bone-mimetic hydrogel can be infilled into the bone layer of the 3D printed structure. The cartilage-mimetic hydrogel with rat MSCs can be infilled into the calcified cartilage layer between the cement line and the tidemark and then above the tidemark. Tire OC-mimetic hydrogel can then be implanted into the OC defect as described in the parent grant for 6 weeks. The repair tissue can be analyzed by (immuno)histochemistry for hyaline cartilage, fibrocartilage, calcified cartilage, and bone. In this embodiment, when the tidemark lattice structure is open or partially polymerized but not degraded, the repair tissue is predominantly hypertrophic. When the tidemark lattice structure is partially polymerized and degraded, the repair tissue exhibits some hypertrophy. On the contrary, when there is a mineralization barrier, the MSCs are protected, allowing them to undergo chondrogenesis from the cues in the hydrogel resulting in predominantly hyaline cartilage with minimal evidence of hypertrophy. REFERENCES

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Claims

CLAIMSWhat is claimed is1. A biomimetic construct comprising a 3D printed structure forming an osteochondral (OC)-mimetic hydrogel scaffold having an independently engineered tidemark-mimetic positioned within the structural support of the hydrogel scaffold.2 The construct of claim 1, further comprising an engineered cement line-mimetic positioned within the structural support of the hydrogel scaffold.3 The construct of claim 1, wherein the tidemark-mimetic restricts the transport of calcifying ions into the articular (hyaline) cartilage layer.

4. The construct of claim 2, wherein the cement line-mimetic prevents cellular migration from the subchondral bone layer into the cartilage layers.

5. The construct of claim 1, wherein the tidemark-mimetic is independently engineered with the scaffold by grayscale patterning, resin chemistry, and / or controlled polymer degradation.

6. The construct of claim 1, wherein the tidemark-mimetic is independently engineered with the scaffold by spatially patterning a degradable resin, such that regions of low conversion partially degrade and produce negatively charged functional groups creating localized regions of negatively charged polymers.

7. The construct of claim 5, wherein the degradable resin comprises a poly(P-amino esters) (PBAE)-acrylate.

8. A method of treating a cartilage injury in a subject, the method comprising placing a therapeutically effective amount of the construct of any of claims 1-7 at the site of the cartilage injury.

9. The method of claim 8, wherein, the cartilage injury is a growth plate injury.

10. The method of claim 9, which prevents the growth of bony bars in cartilage tissue at the site of growth plate injury.

11. The method of claim 9, which treats or prevents the arrest of bone growth at the site of growth plate injury.

12. The method of claim 9, which treats or prevents bone deformities at the site of growth plate injury.

13. The method of claim 9, wherein the subject is an infant, toddler, child, juvenile, adolescent, or young adult.

14. The method of claim 9, wherein the subject is a mammal.

15. The method of claim 14, wherein the subject is a human.

16. A pharmaceutical composition comprising a biomimetic construct comprising a 3D printed structure comprising an osteochondral (OC)-mimetic hydrogel scaffold having an independently engineered tidemark-mimetic positioned within the structural support of the hydrogel scaffold, and a pharmaceutically acceptable carrier.

17. The pharmaceutical composition of claim 1, further comprising an engineered cement linemimetic positioned within the structural support of the hydrogel scaffold.

18. A kit containing the pharmaceutical composition of claim 16 or 17, a container to hold the composition, and a means for administering a therapeutically effective amount of the composition.

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