Composite material for bone regeneration

Abstract

Disclosed herein are scaffolds for osteogenesis comprising a first component and a second component, wherein the first component comprises mineralized collagen glycosaminoglycan (MC-GAG), and the second component comprises poly(D,L-lactide) (PDLLA). Also disclosed herein are methods of promoting osteogenesis and treating bone defects in a subject in need thereof.

Description

[0001] Attorney Docket No. : UCH-42225

[0002] UCLA Ref. No.: [UCLA 2025-048-2] WO

[0003] COMPOSITE MATERIAL FOR BONE REGENERATION

[0004] CROSS-REFERENCE TO RELATED APPLICATIONS

[0005] This patent application claims priority to U.S. Provisional Patent Application No. 63 / 718,088, filed November 8, 2024, which is hereby incorporated by reference in its entirety.

[0006] GOVERNMENT SUPPORT

[0007] This invention was made with government support under DE028098, DE030491, and DE029234 awarded by the National Institutes of Health. The government has certain rights in the invention.

[0008] BACKGROUND

[0009] Cranial defects arising from congenital anomalies, neurosurgical interventions, tumors, trauma, and infections often necessitate cranioplasties. These procedures are crucial for restoring neurological function, ensuring cerebral protection, and improving psychosocial well-being. The current available reconstructive strategies, however, are rife with limitations. Autologous bone grafting is constrained by limited availability and donor site morbidity. Alloplastic materials present an alternative option, albeit with high complication and explantation rates. Consequently, there is an unmet clinical need for regenerative bone biomaterials capable of promoting cranial defect reconstructions in a manner that recapitulates the structure and function of native bone tissue.

[0010] SUMMARY

[0011] In one aspect, the present disclosure provides scaffolds for osteogenesis. In some embodiments, the scaffolds for osteogenesis comprise a first component and a second component, wherein the first component comprises mineralized collagen glycosaminoglycan (MC-GAG), and the second component comprises poly(D,L-lactide) (PDLLA).

[0012] In another aspect, the present disclosure provides methods of promoting osteogenesis in a subject, comprising contacting a scaffold disclosed herein with the bone of the subject.

[0013] In another aspect, the present disclosure provides methods of promoting osteogenesis after injury to a bone in a subject in need thereof, comprising contacting a scaffold disclosed herein with the bone of the subject. Attorney Docket No. : UCH-42225

[0014] UCLA Ref. No.: [UCLA 2025-048-2] WO

[0015] In yet another aspect, the present disclosure provides methods of treating a bone defect in a subject, comprising contacting a scaffold disclosed herein with the bone defect.

[0016] BRIEF DESCRIPTION OF THE FIGURES

[0017] FIG. 1A shows a critical -si zed rabbit cranial defect model that was used to compare the differences in bone healing between unreconstructed defects (Defect only), defects reconstructed with PDLLA implants (Defect-PDLLA), defects reconstructed with MC-GAG scaffolds (MC-GAG), and defects reconstructed with MC-GAG and PDLLA implants (MC- GAG-PDLLA). FIG. IB shows images of each treatment condition initially, at 3 months, and at 9 months.

[0018] FIG. 2A shows ex vivo microCT scanning of the explanted skulls at 3 months. FIG. 2B shows a quantification of the results of FIG. 2A. FIG. 2C shows histologic analyses of explanted defects at the interface between native bone and defect at 3 months. Differences between samples were compared with a Kruskal-Wallis test due to non-normal distributions of the data. Pairwise comparisons were performed using Dunn’s test with a Bonferroni adjustment. A p-value less than 0.05 was considered significant and is marked with three stars (***).

[0019] FIG. 3A shows a measure of strength of the defects, reference point indentation with total indentation distance (TID) at 3 months. FIG. 3B shows a measure of strength of the defects, reference point indentation with the initial cycle indentation distance (ID 1st) at 3 months. FIG. 3C shows a measure of stiffness of the defects, the average relative unloading slope (US) at 3 months. FIG. 3D shows a measure of stiffness of the defects, loading slope (LS) at 3 months. FIG. 3E shows scanning electron microscopy (SEM) images of the surface of the regenerated bone at 3 months. Differences between samples were compared with a Kruskal-Wallis test due to non-normal distributions of the data. Pairwise comparisons were performed using Dunn’s test with a Bonferroni adjustment. A p-value less than 0.05 was considered significant and is marked with three stars (***).

[0020] FIG. 4A shows longitudinal in vivo rabbit skull CT scanning that was conducted at 3, 6, and 9 months. FIG. 4B shows quantification of bone mass, determined as the product of bone volume and mean bone density at each time point based on the CT scans from FIG. 4A.

[0021] FIG. 5A shows a measure of strength of the defects, reference point indentation with total indentation distance (TID) at 9 months. FIG. 5B shows a measure of strength of the Attorney Docket No. : UCH-42225

[0022] UCLA Ref. No.: [UCLA 2025-048-2] WO defects, reference point indentation, with the initial cycle indentation distance (ID 1st) at 9 months. FIG. 5C shows a measure of stiffness of the defects, the average relative unloading slope (US) at 9 months. FIG. 5D shows a measure of stiffness of the defects, loading slope (LS) at 9 months. Differences between samples were compared with a Kruskal-Wallis test due to non-normal distributions of the data. Pairwise comparisons were performed using Dunn’s test with a Bonferroni adjustment. A p-value less than 0.05 was considered significant and is marked with three stars (***).

[0023] DETAILED DESCRIPTION

[0024] In the case of cranial defects, cranioplasties are essential for cerebral protection and neurological restoration, but current materials have limitations, necessitating alternative bone biomaterials for improved outcomes. Therefore, a clinical demand exists for bone biomaterials that mirror tissue-specific extracellular matrix (ECM) properties and regulate progenitor cell fate. Nanoparticulate mineralized collagen glycosaminoglycan (MC-GAG) scaffolds have been demonstrated to promote skull regeneration in vivo without addition of growth factors or exogenous progenitor cells, offering a materials-only solution for cranial defect reconstructions. Further enhancement of the safety and regenerative potential of MC- GAG is however necessary for clinical translation. The present disclosure shows the regenerative effect of MC-GAG scaffold fixation with a resorbable poly(D,L-lactide) (PDLLA) implant for cerebral protection. Fixation of MC-GAG scaffolds with PDLLA implants improved mineralization and bone regeneration compared to either material alone for cranial defect reconstruction.

[0025] Scaffolds

[0026] In one aspect, the present disclosures provides scaffolds for osteogenesis. In some embodiments, the scaffold comprises a first component, wherein the first component comprises mineralized collagen glycosaminoglycan. In some embodiments, collagen and glycosaminoglycan are combined and subjected to a mineralization process, resulting in mineralized collagen glycosaminoglycan. In some embodiments the mineralized collagen glycosaminoglycan comprises microfibrillar type I collagen. In some embodiments the mineralized collagen glycosaminoglycan comprises chondroitin sulfate (e.g., chondroitin-6- sulfate, chondroitin-4-sulfate, chondroitin-4, 6-sulfate, chondroitin-2, 6-sulfate). In some embodiments, the mineralized collagen glycosaminoglycan comprises chondroitin-6-sulfate. Attorney Docket No. : UCH-42225

[0027] UCLA Ref. No.: [UCLA 2025-048-2] WO

[0028] In some embodiments, the mineralized collagen glycosaminoglycan comprises heparan sulfate. In some embodiments, the mineralized collagen glycosaminoglycan comprises hyaluronic acid. In some embodiments, the mineralized collagen glycosaminoglycan comprises at least one, at least two of, or all three of chondroitin sulfate, heparan sulfate, and hyaluronic acid. In some embodiments, the mineralized collagen glycosaminoglycan is crosslinked (e.g, with a water-soluble carbodiimide such as ED AC). In some embodiments, the mineralized collagen glycosaminoglycan is nanoparticulate. In some embodiments, a substance is nanoparticulate if it has particle sizes with an average diameter of approximately 500 nm.

[0029] In some embodiments, the scaffolds comprise a second component, wherein the second component comprises a resorbable polymer, a ceramic (e.g., hydroxyapatite), and / or a metal (e.g., titanium, steel). In some embodiments, resorbable polymer comprises polylactide, poly-glycolide, and / or poly-galactide (e.g., poly-L-lactide, poly-D-lactide, poly-L- glycolide, poly-D-glycolide, poly-L-galactide, and / or poly(D,L-lactide)). In some embodiments, the scaffolds comprise a second component, wherein the second component comprises poly(D,L-lactide).

[0030] In some embodiments, the scaffolds for osteogenesis comprise a first component and a second component, wherein the first component comprises mineralized collagen glycosaminoglycan, and the second component comprises poly(D,L-lactide).

[0031] In some embodiments the scaffold is press-fitted to a bone defect (e.g., a bone defect caused by a surgical wound, a congenital disorder, a cancer, or an infection). In some embodiments, the first component of the scaffold conforms to the bone defect (e.g., as induced via press-fitting). In some embodiments the first component is fixated by the second component. In some embodiments, the second component provides stable and dynamic fixation for the entire scaffold. In some embodiments the second component contains one or more holes each configured to receive a fixing component e.g., a screw or a pin). Screws and pins can be inserted into the holes. In some embodiments the scaffold is fixated with screws. In some embodiments the scaffold is fixated with pins. In some embodiments the fixing component comprises PDLLA.

[0032] In some embodiments, the second component is a PDLLA-based fixator plate (e.g., the ResorbX PDLLA-based fixator plate). In some embodiments, ultrasound-aided fixation (e.g., Sonic Weld Rx) is used for the insertion of pins or screws (e.g., Sonic Pin Rx PDLLA pins). In some embodiments, ultrasound-aided fixation improves fixation stability. In some Attorney Docket No. : UCH-42225

[0033] UCLA Ref. No.: [UCLA 2025-048-2] WO embodiments, ultrasound-aided fixation results in simultaneous welding of the plate, screw or pin, and bone.

[0034] In some embodiments, the scaffolds disclosed herein prevent bone loss. In some embodiments, the scaffolds disclosed herein promote new bone deposition. In some embodiments, the new bone deposition has about the same thickness, strength, and / or stiffness of surrounding native bone.

[0035] In some embodiments, the scaffolds disclosed herein ideally comprise materials with one or more of the following features: ease of handling, proper cell and tissue ingrowth, resorbability, biodegradability, osteoconductivity, stability at the defects site, and adequate mechanical strength throughout the healing process. In some embodiments, the scaffolds prevent stress shielding, alleviate mechanical strain, improve stability, and / or protect against external forces.

[0036] Methods of Treatment

[0037] In another aspect, the present disclosure provides methods of promoting osteogenesis in a subject, the methods comprising contacting a scaffold disclosed herein with the bone of the subject.

[0038] In another aspect, the present disclosure provides methods of promoting osteogenesis after injury to a bone in a subject in need thereof, the methods comprising contacting a scaffold disclosed herein with the bone of the subject.

[0039] In yet another aspect, the present disclosure provides methods of treating a bone defect in a subject, the methods comprising contacting a scaffold disclosed herein with the bone defect.

[0040] In some embodiments, the contacting comprises securing the scaffold to the bone of the subject. In some embodiments, the securing comprises fixation. In some embodiments, the securing comprises press-fitting.

[0041] In some embodiments, the subject has a bone defect. In some embodiments, the subject has a cartilage defect. The bone defect and / or cartilage defect may arise from, for example, a congenital anomaly, a neurosurgical intervention, a tumor, trauma, or an infection. In some embodiments, the subject has a traumatic bone injury. In some embodiments, the subject has an innate bone defect (e.g., a congenital bone defect). In some embodiments, the subject has a surgical wound (e.g., a wound in bone, cartilage). In some embodiments, the Attorney Docket No. : UCH-42225

[0042] UCLA Ref. No.: [UCLA 2025-048-2] WO subject has received a decompressive craniectomy. In some embodiments, the decompressive craniectomy occurred after a stroke and / or cerebral edema (i.e., brain swelling).

[0043] In some embodiments, the method comprises inserting the scaffold into craniofacial bone.

[0044] In some embodiments, the method comprises conjoint administration of one or more agents that prevent infection or promote osteogenesis. In some embodiments the method comprises administering one or more antimicrobial agents (e.g., antibiotics) to the subject.

[0045] In some embodiments, osteogenesis is measurable after about 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, or 12 months. Osteogenesis may be measured by numerous techniques known in the art, such as measuring the strength of the bone (e.g., reference point indentation with total indentation distance (TID), reference point indentation with initial cycle indentation distance (ID 1 st)), measuring the stiffness of the bone (e.g., loading slope (LS), average relative unloading slope (US)), performing histology, performing push tests, and performing imaging (e.g., CT scanning, microCT scanning, scanning electron microscopy (SEM)).

[0046] Definitions

[0047] As used herein, the term “about” is defined as being “ close to” as understood by one of ordinary skill in the art. In one non-limiting embodiment, the term “about” is defined to be within 10%, preferably within 5%, more preferably within 1%, and most preferably within 0.5%.

[0048] As used herein, the term “bone” is understood in the art to refer generally to bone or osseous tissue.

[0049] As used herein, the phrase “conjoint administration” refers to any form of administration of two or more different therapeutic compounds such that the second compound is administered concurrently with the first compound or while the first compound is still effective in the body (e.g., the two compounds are simultaneously effective in the subject, which may include synergistic effects of the two compounds). For example, the different therapeutic compounds can be administered either in the same formulation or in a separate formulation, either concomitantly or sequentially. In certain embodiments, the different therapeutic compounds can be administered within one hour, 12 hours, 24 hours, 36 hours, 48 hours, 72 hours, or a week of one another. Thus, a subject who receives such treatment can benefit from a combined effect of different therapeutic compounds. Attorney Docket No. : UCH-42225

[0050] UCLA Ref. No.: [UCLA 2025-048-2] WO

[0051] As used herein, the terms “fixation", “fixated) and derivatives thereof include the process of stabilizing and securing a scaffold or implant. The scaffold or implant can be secured within a defect, wound, or injury site. In some embodiments, securing the scaffold or implant uses mechanical methods (e.g., screws, pins, plates, rods). In some embodiments, fixation promotes or increases the structural integrity (e.g., load-bearing capacity) of healthy, wounded, or defective bone within and around a scaffold or implant. In some embodiments, a scaffold is fixated to a bone (e.g., on around, or within a cranial defect). In some embodiments, one component of a scaffold is fixated, securing one or more other components of the scaffold in place.

[0052] As used herein, a “fixing component or a “fixating component" is any object that can be used to secure a scaffold to a bone. Screws, pins, plates, mesh, and glue are all examples of fixing components. In some embodiments, fixing components comprise one or more of metal (e.g., steel, titanium), ceramics (e.g., hydroxyapatite), and polymers (e.g., PDLLA). In some embodiments, fixing components are biodegradable and / or resorbable.

[0053] As used herein, the terms “glycosaminoglycan" or “GAG" refer to unbranched polysaccharides. Glycosaminoglycans comprise disaccharide motifs, each comprising an amino sugar (e.g., N-acetyl glucosamine, N-acetyl galactosamine). The disaccharide motif also comprises a uronic acid (e.g., glucuronic acid) or galactose. Examples of glycosaminoglycans include chondroitin sulfate (e.g., chondroitin-6-sulfate, chondroitin-4- sulfate), hyaluronan, heparin, heparan sulfate, dermatan sulfate, and keratan sulfate. Glycosaminoglycans can vary in chain length, sulfation pattern, and molecular weight (e.g., about 20 kDa, about 30 kDa, about 40 kDa, about 50 kDa, about 60 kDa, about 70 kDa, about 80 kDa, about 90 kDa, about 100 kDa, about 110 kDa, about 120 kDa, about 150 kDa, about 200 kDa).

[0054] As used herein, the term “nanoparticulate" refers to a material comprised of nanoparticles. In some embodiments the nanoparticles have a diameter in the range of 100 to 200 nm.

[0055] As used herein, the term “osteogenesis" is understood in the art to refer generally to the formation and / or development of bone.

[0056] As used herein, the terms “PDLLA" , “poly(D,L-lactide)" , or “poly(D,L-lactic acid)" refer to the product of polymerization of a racemic mixture of L-lactides and D-lactides. The PDLLA can be biodegradable and / or resorbable. The ratio of L-lactide to D-lactide in the Attorney Docket No. : UCH-42225

[0057] UCLA Ref. No.: [UCLA 2025-048-2] WO

[0058] PDLLA can vary e.g., about 9: 1, about 8:2, about 7:3, about 6:4, about 5:5, about 4:6, about 3:7, about 2:8, about 1 :9).

[0059] As used herein, a therapeutic that "prevents" a disorder or condition refers to a compound or composition that, in a statistical sample, reduces the occurrence of the disorder or condition in the treated sample relative to an untreated control sample, or delays the onset or reduces the severity of one or more symptoms of the disorder or condition relative to the untreated control sample.

[0060] As used herein, the term “ scaffold ' refers to a three-dimensional object. The scaffold can be implanted (z.e., the scaffold can be an implant). The scaffold can be designed to support the growth or regeneration (z.e., osteogenesis) of bone. In some embodiments, the scaffold can be biodegradable and / or resorbable. For example, in some embodiments, the scaffold can biodegrade within months or years following implantation (e.g., 3 months, 6 months, 9 months, 12 months, or 18 months). In some embodiments, the scaffold can mimic the natural extracellular matrix of bone. In some embodiments, the scaffold can facilitate cell (e.g., osteoblasts, osteocytes, mesenchymal stem cells (MSCs), chondrocytes) attachment, proliferation, or differentiation. In some embodiments, the scaffold comprises biocompatible materials (e.g., polymers, ceramics). In some embodiments, the scaffold can promote vascularization. In some embodiments, the scaffold can promote the integration of new bone with existing bone.

[0061] The term “subject” to which administration is contemplated includes, but is not limited to, humans (z.e., a male or female of any age group, e.g., a pediatric subject (e.g., infant, child, adolescent) or adult subject (e.g., young adult, middle-aged adult or senior adult)) and / or other primates (e.g., cynomolgus monkeys, rhesus monkeys); and / or mammals, including commercially relevant mammals such as cattle, pigs, horses, sheep, goats, cats, and / or dogs. Preferred subjects are humans.

[0062] The term “treating includes prophylactic and / or therapeutic treatments. The term “prophylactic or therapeutic” treatment is art-recognized and includes administration to the subject of one or more of the disclosed compositions (e.g., scaffolds). If it is administered prior to clinical manifestation of the unwanted condition (e.g., disease or other unwanted state of the subject) then the treatment is prophylactic (z.e., it protects the subject against developing the unwanted condition), whereas if it is administered after manifestation of the unwanted condition, the treatment is therapeutic (z.e., it is intended to diminish, ameliorate, or stabilize the existing unwanted condition or side effects thereof). Attorney Docket No. : UCH-42225

[0063] UCLA Ref. No.: [UCLA 2025-048-2] WO

[0064] EXAMPLES

[0065] Example 1: Ultrasonic Fixation of Nanoparticulate Mineralized Collagen Scaffolds with Poly(D.,L-lactide) Implants Improves In Vivo Skull Regeneration

[0066] Summary

[0067] The regenerative effect of mineralized collagen glycosaminoglycan (MC-GAG) scaffold fixation with a resorbable poly(D,L-lactide) (PDLLA) implant for cerebral protection was investigated.

[0068] Fourteen-millimeter cranial defects were created in New Zealand white rabbits, divided into four groups: 1) defect only, 2) defect with PDLLA, 3) MC-GAG, and 4) MC- GAG with PDLLA. Initial bone healing assessment was conducted at 3 months using microcomputed tomography (microCT), histology, reference point indentation, and scanning electron microscopy. Long-term effects were evaluated through in vivo microCT imaging at 3, 6, and 9 months, with biomechanical testing of explanted skulls at 9 months.

[0069] At 3 months, MC-GAG significantly enhanced mineralization compared to empty and PDLLA-treated defects, with even greater mineralization observed for MC-GAG-PDLLA. Histologically, MC-GAG demonstrated trabecular mineralization, while MC-GAG-PDLLA had a structural organization resembling native bone. Biomechanical assessment showed enhanced toughness and stiffness for both MC-GAG and MC-GAG-PDLLA. At 6 months, MC-GAG-PDLLA displayed the highest bone mass, and at 9 months, both MC-GAG- containing scaffolds demonstrated superior bone regeneration compared to controls. By 9 months, PDLLA implants were completely resorbed and MC-GAG-PDLLA exhibited mechanical properties surpassing those of all other groups.

[0070] Fixation of MC-GAG scaffolds with PDLLA implants improved mineralization and bone regeneration compared to either material alone for cranial defect reconstruction.

[0071] Introduction

[0072] While numerous studies have explored the combination of stem cells, growth factors, and scaffolding materials to enhance bone regeneration, none of these strategies have yet translated into clinical practice due to their high costs, long processing times, and unintended side effects. An innovative solution lies in extracellular matrix(ECM)-inspired biomaterials, which harness the instructive capabilities of the ECM to stimulate the regeneration of native Attorney Docket No. : UCH-42225

[0073] UCLA Ref. No.: [UCLA 2025-048-2] WO bone. This emerging strategy holds promise as a safe and viable option for cranial defect reconstruction.

[0074] A nanoparticulate mineralized collagen glycosaminoglycan (MC-GAG) material is effective as a cell-free and growth factor-free strategy for stimulating regeneration of rabbit calvarial bone defects. However, further refinement of the material is essential to enhance both safety and regenerative capacity in preparation for clinical translation. A pertinent challenge faced in the clinical implementation is the protection of underlying structures while allowing adequate intracranial pressure to maintain neurological functioning during early bone healing. To address this challenge, the present example demonstrates how the regenerative capacity of MC-GAG may be influenced by its fixation and overlay with a clinically available resorbable poly-D,L-lactide (PDLLA) implant intended for early-stage cerebral protection.

[0075] Results

[0076] A critical-sized rabbit cranial defect model was used to compare the differences in bone healing between unreconstructed defects (Defect only), defects reconstructed with PDLLA implants (Defect-PDLLA), defects reconstructed with MC-GAG scaffolds (MC- GAG), and defects reconstructed with MC-GAG and PDLLA implants (MC-GAG-PDLLA), as shown in FIG. 1. Initial bone healing assessment was conducted on explanted skulls at 3 months. For a comprehensive understanding of long-term effects, in vivo microCT imaging was subsequently performed at 3, 6, and 9 months, with further examination of explanted skulls at 9 months.

[0077] Mineralization, mechanical, and morphological properties of rabbit calvarial defects reconstructed with MC-GAG and PDLLA implants at 3 months

[0078] At 3 months, the PDLLA implants were not degraded. Ex vivo microCT scanning of the explanted skulls was conducted to analyze mineralized content within the defect at this early timepoint (FIG. 2A). A qualitative improvement in mineralization was observed in skulls reconstructed with MC-GAG, both with and without PDLLA implants. Mineralized content was quantified using median gray values from the microCT scans within a fixed volume of the defect, internally corrected by the density within a fixed volume in the native calvarium for each animal (Defect / Native mineralization ratios; FIG. 2B). Comparisons of Defect / Native mineralization ratios revealed significant differences between groups [H(3)=332.20, pO.OOl], Both MC-GAG [median(IQR): 0.43(1.41)] and MC-GAG-PDLLA Attorney Docket No. : UCH-42225

[0079] UCLA Ref. No.: [UCLA 2025-048-2] WO

[0080] [median(IQR): 0.75(2.04)] exhibited significantly higher mineralization ratios compared to unreconstructed defects [median(IQR): 0.31(0.17), pO.OOOl]. The addition of a PDLLA implant also led to significantly higher mineralization; unreconstructed defects with PDLLA implants [median(IQR): 0.76(0.36)] showed higher ratios than defects only (p<0.0001). Similarly, mineralization ratios were significantly higher in MC-GAG-PDLLA compared to MC-GAG alone (pO.OOl).

[0081] To confirm the presence of mineralization, histologic analyses of explanted defects were performed at the interface between native bone and defect (FIG. 2C). In the unreconstructed defect, predominantly fibrous, non-mineralized soft tissue was observed. In contrast, defects reconstructed with MC-GAG exhibited a more intricate network of trabecular, mineralized content. MC-GAG-PDLLA reconstructions also demonstrated more regenerated bone compared to unreconstructed defects. The mineral content in MC-GAG- reconstructed defects appeared less structurally organized compared to MC-GAG-PDLLA. The latter displayed an organization closely resembling that of the surrounding native bone.

[0082] Next, the mechanical properties of the defect were assessed using reference point indentation with two indicators of strength, namely the initial cycle indentation distance (ID 1st; FIG. 3B) and total indentation distance (TID; FIG. 3A), along with two measures of stiffness, represented by the average relative unloading slope (US; FIG. 3C) and loading slope (LS; FIG. 3D). To mitigate variances in bone healing and thickness across animals, all bioindentation measurements were normalized as relative ratios between the defect and native bone.

[0083] The total indentation distance (TID), which is inversely correlated to microfracture resistance, was found to be different among the groups [H(3)=69.80, p<0.001]. Both MC- GAG [median(IQR): 2.13(1.19)] and MC-GAG-PDLLA [median(IQR): 2.10(1.97)] demonstrated lower TID compared to defect only [median(IQR): 3.09(1.66), p<0.0001 and p=0.03, respectively] and defect-PDLLA controls [median(IQR): 4.63(0.78), pO.OOOl]. The 1stcycle indentation distance (IDlst), which is inversely correlated to mineralization and density, was also found to be significantly different among the groups [H(3)=69.58, pO.OOl], MC-GAG [median(IQR): 2.06(1.16)] and MC-GAG-PDLLA [median(IQR): 2.18(1.92)] exhibited significantly lower relative IDlst values compared to defect-PDLLA controls [median(IQR): 4.57(0.93)], and MC-GAG alone was also significantly lower than defect only controls [median(IQR): 2.90(1.53), pO.OOOl]. No differences were found in Attorney Docket No. : UCH-42225

[0084] UCLA Ref. No.: [UCLA 2025-048-2] WO relative TID and ID 1st values between MC-GAG and MC-GAG-PDLLA (p=0.456 and p=0.125).

[0085] Average relative unloading slope (US) and loading slope (LS) were also found to be significantly different among the groups [H(3)=130.76, pO.OOl; H(3)=123.63, pO.OOl, respectively]. In both measurements, MC-GAG [US median(IQR): 0.34(0.39) and LS median(IQR): 0.34(0.46)], and MC-GAG-PDLLA [US median(IQR): 0.78(0.38) and LS median(IQR): 0.64(0.46)] were significantly higher than defect only [US median(IQR): 0.20(0.19) and LS median(IQR): 0.19(0.24)] and defect-PDLLA controls [US median(IQR): 0.09(0.03) and LS median(IQR): 0.09(0.03), pO.OOl]. MC-GAG-PDLLA also demonstrated higher US and LS values compared to MC-GAG (pO.OOl).

[0086] To evaluate the morphological characterization of native bone, defect only, defect- PDLLA, MC-GAG, and MC-GAG-PDLLA at 3 months, scanning electron microscopy (SEM) images were taken, as shown in FIG. 3E. The surface appearance of the regenerated bone was shown in Figure 3E. Notably, MC-GAG-PDLLA demonstrated a similar interconnected porous structure and composition compared to native bone, whereas this was not visible in the defect only, defect-PDLLA and MC-GAG groups.

[0087] In combination, the micro-CT, histologic, mechanical, and morphological analyses indicate that the combination of MC-GAG with PDLLA was more efficient at mineralization compared to MC-GAG scaffolds.

[0088] Long-term outcomes of rabbit calvarial defects reconstructed with MC-GAG and PDLLA implants

[0089] To assess the long-term effects of MC-GAG-PDLLA on mineralization, longitudinal in vivo rabbit skull CT scanning was conducted at 3, 6, and 9 months (FIG. 4A-B). Initially, no significant differences in bone mass, determined as the product of bone volume and mean bone density, were observed between groups at 3 months (F(3,10)=2.99, p=0.082). At 6 months, there was a significant difference in bone mass between groups [F(3,10)=8.07, p=0.005], with higher values in MC-GAG-PDLLA compared to all other groups. Similarly, at 9 months, significant differences in bone mass were evident between groups [F(3,10)=7.52, p=0.006], with a significantly higher bone mass in MC-GAG-PDLLA compared to both the defect only and defect-PDLLA controls. However, there was no statistical difference between MC-GAG and MC-GAG-PDLLA at 9 months. Attorney Docket No. : UCH-42225

[0090] UCLA Ref. No.: [UCLA 2025-048-2] WO

[0091] The explanted skulls at 9 months demonstrated a complete resolution of the PDLLA implants and were further evaluated for mechanical properties. Both the TID and ID 1st (FIG. 5A-B) were found to be different among the groups [H(3)=38.972, p<0.001 and H(3)=34.907, p<0.001, respectively], MC-GAG-PDLLA consistently exhibited lower relative values for these measurements compared to all other groups. Similarly, the stiffness measurements, US and LS (FIG. 5C-D), demonstrated significant differences between the groups [H(3)=54.531, p<0.001 and H(3)=53.952, p<0.001, respectively], with higher values in MC-GAG-PDLLA compared to all other groups.

[0092] Taken together, these data indicate that MC-GAG-PDLLA promoted enhanced mineralization at 6 months and exhibited improved mechanical properties at 9 months compared to all other groups.

[0093] Conclusion

[0094] Rabbit cranial defects were induced and reconstructed with an MC-GAG scaffolds stabilized by a Poly(D,L-lactide) (PDLLA) plate onlay fixed with PDLLA screws, and were compared to empty defects without treatment, defects reconstructed with a press-fitted MC- GAG scaffold alone, and unreconstructed defects treated with the PDLLA onlay implant alone. The explanted skulls were assessed at 3 and 9 months following implantation, with in vivo microCT analyses conducted at 3, 6, and 9 months. At 3 months, the explanted skulls revealed the MC-GAG scaffold promoted mineralization in comparison to the PDLLA- treated and empty defects, with even greater mineralization observed for the MC-GAG- PDLLA combination scaffold. Histological analysis of the interface between the reconstruction and native bone revealed that MC-GAG promoted the development of an intricate network of trabecular mineralized content compared to the untreated defect — primarily featuring fibrous, non-mineralized tissue — with enhanced structural organization more closely representative of nearby native bone for MC-GAG-PDLLA. Examining the biomechanical properties of the treated skulls revealed enhanced hardness and microfracture resistance of both MC-GAG alone and MC-GAG-PDLLA compared to the untreated and PDLLA-treated defects, with significantly greater measures of stiffness for MC-GAG- PDLLA. While no difference in regenerated bone mass measured by in vivo CT was detected between the treatment groups after 3 months, MC-GAG-PDLLA featured the greatest bone mass at 6 months, and both MC-GAG-containing scaffolds demonstrated enhanced bone regeneration compared to the PDLLA and empty defects at 9 months. While the PDLLA Attorney Docket No. : UCH-42225

[0095] UCLA Ref. No.: [UCLA 2025-048-2] WO plates were still discernible at the 3-month mark, they were completely resorbed by 9 months. Analyses of the mechanical properties of explanted skulls after 9 months revealed that both strength and stiffness were greatest and most similar to native bone for MC-GAG-PDLLA. Together, these results demonstrate that the reconstruction of a bone defect with a MC-GAG scaffold fixated with PDLLA significantly enhanced long-term mineralization and bone regeneration compared to a scaffold consisting of either MC-GAG or PDLLA alone.

[0096] Example 2. Methods and Materials used in Example 1

[0097] Fabrication and chemical crosslinking of non-mineralized and mineralized collagen scaffolds Nanoparticulate mineralized collagen glycosaminoglycan (MC-GAG) scaffolds were fabricated through lyophilization as described in Weisgerber et al. “The impact of discrete compartments of a multi-compartment collagen-GAG scaffold on overall construct biophysical properties.” J Meeh Behav Biomed Mater. 2013 Dec; 28:26-36. Briefly, a mixture of 0.5 w / w% microfibrillar type I collagen from bovine Achilles tendon (Collagen Matrix, Oakland, NJ) and 0.044 w / w% chondroitin-6-sulfate from shark cartilage (Sigma- Aldrich, St. Louis, MO) was combined in a phosphoric acid solution containing calcium salts (calcium nitrate hydrate: Ca(NO3)2 4H2O; calcium hydroxide: Ca(OH)2, Sigma-Aldrich; further details can be found in Lynn & Bonfield “A novel method for the simultaneous, titrant-free control of pH and Calcium Phosphate mass yield” Accounts of Chemical Research, 2005; 38(3), 202-207). The solution was gradually frozen at a rate of 1 °C / min using a freeze dryer (Genesis, VirTis, Gardiner, NY), transitioning from room temperature to -10 °C. After sublimation, the scaffolds underwent sterilization with ethylene oxide and were stored at -20 °C. Scaffolds were cut into 14 mm disks and rehydrated with ethanol, followed by phosphate-buffered saline (PBS) overnight. Subsequently, the scaffolds underwent crosslinking using l-ethyl-3 -(3 -dimethylaminopropyl) carbodiimide (EDAC, Sigma-Aldrich) and N-hydroxysuccinimide (NHS, Sigma-Aldrich), combined at room temperature for 4 hours at a pH of 5.5, as described in Olde Damink et al. “Cross-linking of dermal sheep collagen using a water-soluble carbodiimide.” Biomaterials. 1996 Apr;17(8):765-73. Other cross-linking reagents and conditions can be used to alter the properties of the scaffold, as demonstrated in Lynn & Bonfield (cited above). To eliminate residual chemicals, the scaffolds were washed and incubated in PBS. Attorney Docket No. : UCH-42225

[0098] UCLA Ref. No.: [UCLA 2025-048-2] WO

[0099] In vivo rabbit cranial defect reconstruction and PDLLA fixation

[0100] Animal experiments were performed in compliance with the USDA Animal Welfare Act and PHS Policy for the Humane Care and Use of Laboratory Animals. Preoperatively, New Zealand White rabbits (2-3 months old; Charles River Laboratories, Wilmington, MA) were given an intravenous injection of 2-5 mg / kg alfaxalone or 0.01 mg / kg dexmedetomidine for induction. The head of each rabbit was shaved and disinfected with Betadine. Anesthesia was maintained with isoflurane gas (1.5-3%) during the procedure and pain control was performed with a subcutaneous injection of 0.02 mg / kg buprenorphine.

[0101] Rabbits were divided into 4 groups: 1) defect without reconstruction, 2) defect with PDLLA implant, 3) MC-GAG scaffold only, and 4) MC-GAG with PDLLA implant. The cranial surface was exposed by a midline incision and the overlying parietal periosteum was dissected off of the calvarium. For each rabbit, a 14-mm biparietal full thickness, extradural defect was created by a hand powered trephine and the bone was lifted away without injury to the dura. One scaffold was implanted for each rabbit. In groups 2 and 4, 22-mm resorbable Poly(D,L-lactide) implants (Resorb x, KLS-Martin L.P., Jacksonville, FL) were fixed with two 3-mm resorbable PDLLA pins (SonicPin Rx, K KLS-Martin L.P.) using SonicWeld Rx advanced ultrasound technology (KLS-Martin L.P.). The incision was closed with 5-0 resorbable Vicryl sutures. Postoperative, rabbits were given 0.03 mg / kg buprenorphine twice- daily for two days, 0.3 mg / kg meloxicam daily for 5 days, and 10 mg / kg orbifloxacin for 10 days.

[0102] Three and nine months after implantation, the rabbits were euthanized by intravenous injection of 1 mL of pentobarbital solution (Fatal-Plus®, 390 mg / ml) intravenously via the marginal ear vein. The previous incision was then reopened and the calvarium was exposed. The calvarium including the cranial defect was analyzed grossly and then explanted for micro-computed tomography, histologic, and biomechanical analyses.

[0103] Micro-Computed Tomographic Imaging

[0104] Ex-vivo rabbit skull micro-computed tomographic imaging (pCT) was performed using the Scanco pCT 35 (Scanco Medical AG, Bruttisellen, Switzerland). Skulls were removed 3 and 9 months after the implantation of scaffolds, fixed in 10% formalin for 24 hours, and then stored in 70% ethanol at 4 °C until scanned. Scans were performed in 70% ethanol using high resolution settings with a source voltage of 45 E (kVp), Intensity (pA) of 177, and a voxel size of 15 pm. Skull areas were contoured to establish volumes of interest Attorney Docket No. : UCH-42225

[0105] UCLA Ref. No.: [UCLA 2025-048-2] WO and an optimum arbitrary threshold value of 415 was used uniformly for all specimens to quantify mineralized bone areas from surrounding unmineralized scaffold. Three-dimensional reconstruction pictures were generated, and the volume of old and new bone calculated. For density calculations, DICOM files were imported into ImageJ (NIH, Bethesda, MD) and mean Hounsfield Units (HU) were obtained from a cylindrical volume of 0.045 cm3consisting of the defect and 0.03 cm3outside of the defect in the native bone as an internal control. The ratio of mean density of defect / mean density of native bone was then calculated for each condition.

[0106] In vivo rabbit cranium imaging was performed at the University of California, Los Angeles (UCLA) Crump Preclinical Imaging Technology Center. During the survival period at 3, 6, and 9 months after implantation, rabbits were injected with 1-5 mg / kg alfaxalone before anesthesia was maintained with 3-5% isoflurane gas. Each rabbit was placed on the imaging bed of a GNEXT Positron Emission Tomography and Computed Tomography (PET / CT) scanner (Sofie Biosciences, Culver City, CA). The rabbit cranium was scanned with a source voltage of 80 kVp, Intensity (pA) of 150, using 720 projections, and a 1 -minute scan time. Three-dimensional (3D) image reconstruction was performed using a Modified Feldkamp Algorithm and analyzed with Dragonfly software (Dragonfly 4.0, Object Research Systems Inc., Montreal, Canada). 3D reconstructions were cropped and a 0.92 cm3cylindrical region of interest (RO I) was placed on the defect. The Otsu segmentation method was used to distinguish bone from non-bone. The regenerated bone mass was subsequently calculated by multiplying the bone volume (mm3) and density (Hounsfield Units, HU) (21,22).

[0107] Histology and immunohistochemistry

[0108] Skulls were removed 3 months after the implantation of scaffolds, fixed in 10% formalin, decalcified, embedded in paraffin, and sectioned at 4 microns using standard techniques. The sections were deparaffinized and stained with hematoxylin and eosin. Images were captured with the Zeiss Axio Observer 3 inverted microscope with the ZEN 2.3 Pro software (Zeiss, Oberkochen, Germany) and analyzed qualitatively.

[0109] Reference point indentation for in vivo rabbit cranial defects

[0110] Explanted rabbit skulls at 3 and 9 months were fixed and tested with the BioDent reference point indentation device (Active Life Scientific, Santa Barbara, CA) according to manufacturer’s instructions. Indentations were conducted in 10 areas of native bone and 20 Attorney Docket No. : UCH-42225

[0111] UCLA Ref. No.: [UCLA 2025-048-2] WO areas of regenerated bone at a force of 2N, an indentation frequency of 2 Hz, and 10 indentation cycles at a touchdown force of 0.1 N using a probe assembly type BP2. Indentation data was analyzed with the BioDent software for the total indentation distance (TID), first cycle indentation distance (IDlst), loading slope (LS), and unloading slopes (US). Toughness, or resistance to fracture, was determined by the total distance of indentation (TID) reached by the test probe and the first cycle indentation distance (IDlst). Relative stiffness was determined by the loading (LS) and unloading (US) slopes of the force (N) to displacement (pm) curves. To minimize differences in the thickness of bone for each animal as well as the bone healing capabilities, data from each cranial defect was internally controlled with the native calvarial bone.

[0112] Scanning Electron Microscopy (SEM) imaging

[0113] Morphological characterization of was visualized using Scanning Electron Microscopy (SEM) (Supra 40VP SEM, ZEISS, Germany). Explanted rabbit skulls at 3 months were fixed in 10% formalin overnight. Next, samples were rinsed with PBS, decalcified using a 0.5 M ethylenediaminetetraacetic acetic (EDTA) disodium salt solution, and stored in 70% ethanol. Subsequently, samples were placed inside a critical -point dryer (AutoSamdri 810 Critical Point Dryer, Tousimis, USA). Dried samples were then coated with a gold target (Ted Pella, USA) using a sputter coater (Pelco SC-7, Ted Pella, USA) and imaged.

[0114] Statistical Analysis

[0115] Differences between ex vivo microCT and reference point indentation analyses were compared with a Kruskal-Wallis test due to non-normal distributions of the data. Pairwise comparisons were performed using Dunn’s test with a Bonferroni adjustment. Mean differences between in vivo microCT analyses were compared with a one-way ANOVA with posthoc comparisons under the Tukey criterion. A p-value less than 0.05 was considered significant. All statistical analyses were performed using SPSS software Version 28 (SPSS, Inc., Chicago, IL). Attorney Docket No. : UCH-42225

[0116] UCLA Ref. No.: [UCLA 2025-048-2] WO

[0117] INCORPORATION BY REFERENCE

[0118] All publications and patents mentioned herein are hereby incorporated by reference in their entirety as if each individual publication or patent was specifically and individually indicated to be incorporated by reference. In case of conflict, the present application, including any definitions herein, will control.

[0119] EQUIVALENTS

[0120] While specific embodiments of the subject invention have been discussed, the above specification is illustrative and not restrictive. Many variations of the invention will become apparent to those skilled in the art upon review of this specification and the claims below. The full scope of the invention should be determined by reference to the claims, along with their full scope of equivalents, and the specification, along with such variations.

Claims

Attorney Docket No. : UCH-42225UCLA Ref. No.: [UCLA 2025-048-2] WOCLAIMSWhat is claimed is:

1. A scaffold for osteogenesis comprising a first component and a second component, wherein the first component comprises mineralized collagen glycosaminoglycan, and the second component comprises poly(D,L-lactide) (PDLLA).

2. The scaffold of claim 1, wherein the mineralized collagen glycosaminoglycan comprises microfibrillar type I collagen.

3. The scaffold of claim 1 or 2, wherein the mineralized collagen glycosaminoglycan comprises chondroitin sulfate.

4. The scaffold of claim 3, wherein the mineralized collagen glycosaminoglycan comprises chondroitin-6-sulfate.

5. The scaffold of any one of claims 1-4, wherein the mineralized collagen glycosaminoglycan is crosslinked with a water-soluble carbodiimide (e.g., ED AC).

6. The scaffold of any one of claims 1-5, wherein the mineralized collagen glycosaminoglycan is nanoparticulate.

7. The scaffold of any one of claims 1-6, wherein the scaffold is press-fitted to a bone defect (e.g., a bone defect caused by a surgical wound, a congenital disorder, a cancer, or an infection).

8. The scaffold of any one of claims 1-7, wherein the first component is fixated by the second component.

9. The scaffold of any one of claims 1-8, wherein the second component contains one or more holes each configured to receive a fixing component (e.g., a screw or a pin).

10. The scaffold of any one of claims 1-9, wherein the scaffold is fixated with screws or pins.

11. The scaffold of claim 9, wherein the fixing component comprises PDLLA.Attorney Docket No. : UCH-42225UCLA Ref. No.: [UCLA 2025-048-2] WO12. A method of promoting osteogenesis in a subject, the method comprising contacting the scaffold of any one of claims 1-11 with a bone of the subject.

13. A method of promoting osteogenesis after injury to a bone in a subject in need thereof, the method comprising contacting the scaffold of any one of claims 1-11 with the bone of the subject.

14. The method of claim 12 or 13, wherein the subject has a traumatic bone injury.

15. The method of any one of claims 12-14, wherein the subject has a bone defect.

16. A method of treating a bone defect in a subject, the method comprising contacting the scaffold of any one of claims 1-11 with the bone defect.

17. The method of any one of claims 12-16, wherein the contacting comprises securing the scaffold to the bone of the subject.

18. The method of claim 17, wherein securing the scaffold to the bone of the subject comprises fixation.

19. The method of claim 17 or 18, wherein securing the scaffold to the bone of the subject comprises press-fitting.

20. The method of any one of claims 12-19, wherein the subject has an innate bone defect (e.g., a congenital bone defect).

21. The method of any one of claims 12-20, wherein the subject has a surgical wound.

22. The method of any one of claims 12-21, wherein the method comprises administering an antimicrobial agent (e.g., an antibiotic) to the subject.

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