Comparison of different 3D-printed allogeneic bone and artificial polymer composite porous scaffold materials for repairing cranial bone defects in rats

Abstract

BACKGROUND: Allogeneic bone repair materials have good effect on bone defect repair, but they have drawbacks such as immune rejection, high cost, and low rigidity. OBJECTIVE: To evaluate the bone repair ability, tissue response and degradation performance of three kinds of 3D-printed allogeneic bone and artificial polymer composite porous scaffold materials. METHODS: Three kinds of bone repair materials were prepared: (1) Sample A: A bone repair material was prepared by mixing polylactic acid-glycolic acid copolymer with allogeneic bone powder at a mass ratio of 1:4, using low-temperature deposition 3D printing. (2) Sample B: A bone repair material was prepared by mixing polycaprolactone with allogeneic bone powder at a mass ratio of 1:4, using low-temperature deposition 3D printing. (3) Sample C: A bone repair material was prepared by mixing polycaprolactone with allogeneic bone powder at a mass ratio of 3:7, using high-temperature melt 3D printing. In 35 SD rats, one 5 mm diameter circular bone defect was drilled on each side of the sagittal suture. Ten defects received no intervention (blank control group), 15 defects were implanted with allogeneic bone material (positive control group), 15 defects were implanted with sample A (sample A group), 15 defects with sample B (sample B group), and 15 defects with sample C (sample C group). At 2, 4, 8, 12, and 26 weeks postoperatively, samples were harvested. Hematoxylin-eosin staining was used to observe material degradation, tissue response, and new bone formation. Masson staining was used to observe collagen fiber formation. Immunohistochemical staining was used to observe the expression of RUNT-related transcription factor 2 and type I collagen. RESULTS AND CONCLUSION: (1) Hematoxylin-eosin staining: During the experimental period, all implanted materials degraded to varying degrees. Samples A and B degraded faster, followed by the positive control, and sample C degraded slower. Over time, the inflammatory response in each implanted material group tended to decrease, but fibrous tissue proliferation and neovascularization were still obvious. At 26 weeks, the inflammatory response in sample A group was significantly reduced, followed by sample B group and positive control group, while sample C group showed no obvious reduction. In sample B group, a small amount of new bone formation was visible at 8 weeks; in sample A and positive control groups, new bone formation was visible at 12 weeks; these three groups showed a large amount of new bone formation at 26 weeks. Sample C group never showed obvious new bone formation. (2) Masson staining: At 2 weeks, a small amount of disordered collagen deposition was seen in each implanted material group; at 26 weeks, a large amount of regularly arranged collagen deposition was seen. (3) Immunohistochemical staining: Over time, the expression of RUNT-related transcription factor 2 and type I collagen increased in each implanted material group. At 12 and 26 weeks, the expression in positive control, sample A, and sample B groups was higher than that in sample C group. (4) These results indicate that samples prepared by low-temperature deposition 3D printing of polylactic acid-glycolic acid copolymer or polycaprolactone with allogeneic bone powder degraded faster, reduced inflammatory response, and showed more obvious expression of osteogenic factors and new bone formation than samples prepared by high-temperature melt printing of high-content polycaprolactone with allogeneic bone powder.

1. Introduction

Cranial defects caused by craniocerebral trauma, tumors, cerebrovascular diseases, and infections often require subsequent cranioplasty [1-2]. The development of bone tissue engineering has opened new avenues for repairing bone defects, aiming to induce functional bone regeneration through the synergistic combination of biomaterials, cells, and growth factors. A key component is the scaffold material that provides structural support for new bone tissue. In recent years, 3D printing technology has provided new methods for personalized repair of cranial defects. Porous scaffold materials fabricated by 3D printing can not only construct complex shapes matching the defect bone tissue but also precisely control the internal pore structure, and can carry bioactive factors and cells for in situ printing at the defect site, thereby achieving ideal bone repair effects [2-3].

Allogeneic bone has good osteoconductive and osteoinductive capabilities and has been used clinically for bone defect treatment [4-5], but it has drawbacks such as immune rejection, high cost, and low rigidity. Polycaprolactone (PCL) and polylactic acid-glycolic acid copolymer (PLGA) are synthetic polymer materials that have been extensively studied due to their good biocompatibility, tunable degradation properties, and mechanical performance, and are widely used in tissue engineering [6-7]. However, they have issues such as poor cell adhesion, lack of bioactivity, and induction of tissue reactions [8]. Therefore, combining different materials to improve bone repair capacity is a new therapeutic strategy for bone defects. However, there is limited research on the repair effect of porous scaffold materials prepared by 3D printing of allogeneic bone combined with artificial polymers. Studies have shown that RUNT-related transcription factor 2 (RUNX2) and type I collagen are essential transcription factors for osteoblast differentiation and play crucial roles in osteoblast development, differentiation, and regulation [9-11]. This study used a rat cranial defect model to evaluate the bone repair ability, tissue response, and degradation performance of three types of 3D-printed composite porous scaffolds made of allogeneic bone and artificial polymers (PLGA or PCL). By observing the expression changes of RUNX2 and type I collagen, the effects of these materials on repairing cranial defects were analyzed.