In vivo research on 3D-printed composite PLGA and PDLLA-HA absorbable scaffolds for repairing radius defects in rabbits

Abstract

Objectives

Despite being an important research topic in oral biomaterials, few studies have demonstrated the differences between poly(d,l-lactide-co-glycolide)/hydroxyapatite (PLGA/HA) and poly(d,l-lactic acid)/hydroxyapatite (PDLLA/HA). In this study, PLGA/HA and PDLLA/HA scaffolds were prepared using three-dimensional (3D) printing technology and implanted into radius defects in rabbits to assess their effects on bone regeneration.

Methods

In this study, 6 mm × 4 mm bone defects were generated in the bilateral radii of rabbits. 3D-printed PLGA/HA and PDLLA/HA scaffolds were implanted into the defects. X-ray imaging, micro-computed tomography, and hematoxylin–eosin staining were performed to observe the degradation of the materials, the presence of new bone, and bone remodeling in the bone defect area.

Results

The PLGA/HA scaffolds displayed complete degradation at 20 weeks, whereas PDLLA/HA scaffolds exhibited incomplete degradation. Active osteoblasts were detected in both groups. The formation of new bone, bone marrow cavity reconstruction, and cortical bone remodeling were better in the PLGA/HA group than in the PDLLA/HA group.

Conclusions

PLGA/HA scaffolds performed better than PDLLA/HA scaffolds in repairing bone defects, making the former scaffolds more suitable as bone substitutes at the same high molecular weight.

Keywords

3D printing hydroxyapatite scaffold bone defect bone regeneration

Introduction

Large numbers of patients develop jaw defects clinically caused by trauma, infection, tumor, congenital malformation, and other reasons, thereby creating a large demand for bone-repairing materials.^1,2 Natural bone tissue is mainly composed of biological apatite and collagen molecules,³ conferring the advantages of mechanical strength and easy tissue regeneration. These properties are attributed to the structure of bone, which forms a matrix with collagen, whereas biological apatite acts as an inorganic phase.^4,5 Recently, bone repair materials mimicking this structure have attracted substantial attention from scholars. These bone repair materials primarily consist of biopolymers and inorganic ceramics.⁶ Biomaterials can be natural (e.g., collagen, hyaluronic acid) or synthetic (e.g., polyester). However, synthetic biomaterials have been widely adopted as bone substitutes in various research and clinical applications because of the shortcomings of natural biomaterials concerning antigenicity, immunogenicity, and availability.⁷

Poly(d,l-lactide-co-glycolide)/hydroxyapatite (PLGA/HA) and poly(d,l-lactic acid)/hydroxyapatite (PDLLA/HA) are two synthetic polymer materials used for tissue engineering.^8,9 However, they are rarely used as monomers because they accumulate acidic products during the degradation process in vivo, resulting in aseptic inflammation.^10,11 HA is an important component of the human endoskeleton.¹² Recently, many scholars have attempted to add HA to polyester materials to neutralize the acidic environment, and successful effects were observed.^13,14 However, the addition of excessive amounts of HA can make it difficult to extrude the material. Prior research revealed that a biocomposite of poly(lactic acid) and 10 wt% HA exhibited strong shear thinning behavior, making it more suitable for fused filament fabrication because lower printing pressure is required.¹⁵ Cui explored the effects of various PLGA and HA ratios on the performance of the scaffold through osteoblast adhesion and radial bone implantation in rabbits. Cell activity, the cell adhesion rate, and bone defect repair were better in the 10 and 20 wt% HA groups than in the 5 and 40 wt% HA groups,¹⁶ demonstrating that a polymer/HA ratio of 9:1 is appropriate for 3D-printed composites. PLGA/HA and PDLLA/HA have good prospects for repairing bone defects. Prior studies demonstrated that 3D-printed PLGA/HA and PDLLA/HA scaffolds have good mechanical properties, in vitro biocompatibility, and in vivo osteogenic ability.^17,18 However, few studies have compared their properties horizontally.¹⁹

In this study, we selected high-molecular-weight PLGA and PDLLA and then added HA to both materials. Next, we prepared scaffolds using 3D printing technology and implanted them into radial defects in rabbits to observe the degradation of scaffolds in vivo and new bone regeneration in the bone defect area. Subsequently, we compared the performance of PLGA/HA and PDLLA/HA by X-ray imaging, micro-computed tomography (CT), and hematoxylin–eosin (HE) staining.

Materials and methods

Scaffold preparation

HA powder (Nanjing Dulai Biomaterial Co., Ltd, Nanjing, China), accounting for 10% of the total mass, was added to PLGA (75:25) and PDLLA material powders (average molecular weight, 300,000; Jinan Daigang Biomaterial Co., Ltd, Jinan, China). The two powders were mixed with HA, poured into a V-type mixer, and then poured into an extruder for 30 minutes of mixing. The temperature of the mixing chamber of the extruder was 199°C, whereas that of the melting section was 195°C, with both temperatures checked after extruding the wires of both materials. The diameter of PLGA-HA and PDLLA-HA wires was 1.65 ± 0.15 mm after removing the badly drawn parts in the wires. A 6-mm × 4-mm × 4-mm scaffold was designed for both polymers and imported into Cura slicing software (Ultimaker, Geldermalsen, Netherlands). After slicing and layering, the printing parameters were a layer height of 0.1 mm, filling rate of 100%, and printing rate of 50 mm/minute. Each drawing-formed wire was loaded into the 3D printer, which had a printer nozzle of 0.4 mm. The design of the printed wire diameter was 500 μm, and the hole diameter was 400 μm. Finally, the printing was begun, and the printed copy was obtained. All PLGA/HA and PDLLA/HA samples (Figure 1) were packaged and sterilized by radiation.

Figure 1.

PLGA/HA and PDLLA/HA scaffolds. PLGA/HA, poly(d,l-lactide-co-glycolide)/hydroxyapatite; PDLLA/HA, poly(d,l-lactic acid)/hydroxyapatite.

Animal model

All experimental procedures and protocols adopted in this research were reviewed and approved by the Animal Care and Ethics Committee of the People’s Hospital of Inner Mongolia Autonomous Region. The study protocol was approved by the Animal Care and Ethics Committee of the People’s Hospital of Inner Mongolia Autonomous Region (No. 2022LL032). We selected nine healthy 3-month-old male New Zealand white rabbits (Xian Dilepu Biomedical Co., Ltd., Xian, China) weighing 2.0 to 2.5 kg. All surgeries were performed under general anesthesia (via an intramuscular injection of 30 mg/kg sodium pentobarbital [Merck, Darmstadt, Germany]). After locally injecting 1 mL of articaine (Produits Dentaires Pierre Rolland, Merignac, France), the experimental rabbits were placed in the supine position, and their limbs were fixated to the operating table. The skin was prepared routinely, disinfected, and covered with a towel. The forearms of rabbits were randomly assigned to the PLGA/HA or PDLLA/HA group. Next, an approximately 5-cm incision was made in the forearms of both rabbits, and the fascia and muscle were separated layer-by-layer to expose the radius. The middle part of the radius, including the periosteum, was excised using a turbine machine (Bien-Air Medical Technologies, Bienne, Switzerland), thereby generating a bone defect of 6 mm × 4 mm ×4 mm. Later, the corresponding scaffolds were implanted in both groups, and the layers in the wound were closely sutured. Rabbits were reared in separate cages and injected intramuscularly with 0.1 mL/kg penicillin (North China Pharmaceutical Co., Ltd., Shijiazhuang, China) to prevent infection. The surgery was performed twice a day for 5 days. Subsequently, the thread was removed after 10 days, and one animal from each group was sacrificed via an overdose of the anesthetic 4, 8, and 20 weeks after the surgery.

Radiography and micro-CT analysis

To evaluate new bone formation, the fracture line, and bone marrow cavity reconstruction at the bone defect, X-ray imaging (IntraOs 70, Fona, Assago, Italy) and micro-CT (Inveon Micro-CT, Siemens, Munich, Germany) were performed on the forearm specimens. The bone volume/tissue volume ratio (BV/TV) and bone mineral density (BMD) at different healing times were calculated to evaluate the degree of osteogenesis.

Histology

Each radius was separated and fixed in 10% neutral buffered formalin solution (Shandong Yilan Biochemical Co., Ltd., Jinan, China). Next, it was decalcified in formic acid and embedded in paraffin to create hard tissue grinding tablets. The specimens were stained with HE and examined histologically under a light microscope to examine the formation of new bone in the bone defect region along with the progression of the scaffold degradation. Image-Pro Plus 6.0 software (Media Cybernetics, Silver Spring, MD, USA) was applied to analyze the residual material area and new bone formation area.

Statistical analyses

SPSS 25.0 software (IBM Corp., Armonk, NY, USA) was used for statistical analysis. The groups were compared using the independent-samples t-test. The difference was considered statistically significant at P < 0.05.

Results

General observations

All experimental rabbits recovered well after the operation and displayed a good appetite. All animals were lively, their wounds did not crack, and none died.

In the PLGA/HA group, the edge of the material was smooth, and the callus formed 4 weeks after the operation. In the PDLLA/HA group, the outline of the material was clear with no obvious callus formation. In the PLGA/HA group, the callus completely surrounded the wound and material 8 weeks after surgery, exhibiting swelling and softness. In the PDLLA/HA group, the edges of the materials were smooth, and the initial callus was formed at this time. In the PLGA/HA group, the material had almost completely degraded at 20 weeks after surgery, and the wound was smooth and hard to the touch. In the PDLLA/HA group, the callus completely surrounded the wound, and the callus was swollen and soft to the touch (Figure 2a).

Figure 2.

Model of bone defects and the results of imaging. (a) Images of the rabbit radius specimens in the PLGA/HA and PDLLA/HA groups at 4, 8, and 20 weeks after surgery. (b) X-ray films of the rabbit radius specimens in the PLGA/HA and PDLLA/HA groups at 4, 8, and 20 weeks after surgery. (c) Micro-CT at 4, 8, and 20 weeks after surgery. (d) BV/TV at 4, 8, and 20 weeks after surgery and (e) BMD at 4, 8, and 20 weeks after surgery. *P > 0.05 compared with the PDLLA/HA group; **P < 0.05 compared with the PDLLA/HA group. PLGA/HA, poly(d,l-lactide-co-glycolide)/hydroxyapatite; PDLLA/HA, poly(d,l-lactic acid)/hydroxyapatite; BV/TV, bone volume/total volume ratio; BMD, bone mineral density.

X-ray examination

In both groups, no significant change was observed in the operation area after 4 weeks. However, at 8 weeks after surgery, the texture of the cut ends was rough in the PLGA/HA group, with the cortical bone at the cut ends extending to the middle and bone components being formed in the defect area. In the PDLLA/HA group, the broken end of the incision was smooth with less bone formation in the middle of the defect. At 20 weeks after surgery, blurred broken ends were observed in the PLGA/HA group, whereas the bone defects were filled with obvious bone components. In the PDLLA/HA group, the broken end of the incision remained visible with more bone components being formed at the bone defect (Figure 2b).

Micro-CT

There was no significant change in the surgical area after 4 weeks in both groups. At 8 weeks after surgery, new bone formation in the defect area was observed in both groups. BV/TV was significantly higher in the PLGA/HA group than in the PDLLA/HA group after 8 weeks (P < 0.05), whereas no difference in BMD was noted between the groups. After 20 weeks, the defect area in the PLGA/HA group was completely filled with new bone, whereas defects remained in the PDLLA/HA group. BV/TV and BMD at 20 weeks after surgery were significantly higher in the PLGA/HA group than in the PDLLA/HA group (both P < 0.05, Figure 2c).

Histological examination

Four weeks after surgery, the operated broken ends in both groups were clear, and internal and external calluses were formed at the radius defect. Moreover, a large number of new bone trabeculae and cartilage islands were found in the callus, in addition to irregularly arranged bone trabeculae. In the PLGA/HA group, the degradation of materials was obvious in the defect cavity together with disordered growth and arrangement of new bone. Osteoblasts were active, and bone marrow-like tissue was present, reflecting the natural bone-healing state. However, the new bone tissue in the PDLLA/HA group extended regularly along the direction of the pore diameter of the material, suggesting slow degradation of the material that hindered the new bone. Eight weeks after surgery, marginal tissues were observed to grow and extend in both groups, with active osteoclasts being observed in the outer callus. As the materials degraded and the internal callus disappeared in the PLGA/HA group, the number of bone marrow-like tissues increased greatly, facilitating the fusion of the bone marrow cavity and defect cavity. However, in the PDLLA/HA group, the broken ends remained visible with fibrous bone growing along the pore diameter of the material. At 20 weeks, obvious marks of the broken ends and the residual contour of the filling materials were not observed in the PLGA/HA group. In addition, the cortical bone was reconstructed and connected, and the cartilage islands were reduced, exhibiting new tabular bone with a regular arrangement. Moreover, the defect was filled with mature bone marrow tissue, whereas the bone marrow cavity was fused with the defect. Although broken ends remained visible in the PDLLA/HA group, the fibrous callus was reduced at the broken ends. Furthermore, the reconstruction of cortical bone was identical to that in the PLGA/HA group, with bone marrow-like tissues observed in the defect together with some fused bone marrow cavities and defects (Figure 3).

Figure 3.

Cross-sectional hematoxylin–eosin staining of specimens in the PLGA/HA and PDLLA/HA groups at 4, 8, and 20 weeks after stent implantation. PLGA/HA, poly(d,l-lactide-co-glycolide)/hydroxyapatite; PDLLA/HA, poly(d,l-lactic acid)/hydroxyapatite; NB, new bone tissue; CB, cartilage tissue; OB, osteoblast; OCL, osteoclast; LNB, plate bone.

Histological scoring

The histology of the repaired defects was compared between the groups at 4, 8, and 20 weeks after surgery, and the area of residual materials and the area of new bone formation were evaluated. No differences were observed between the two groups at 4 weeks after surgery. However, after 8 and 20 weeks, the histological scores were significantly better in the PLGA/HA group than in the PDLLA/HA group (P < 0.05, Figure 4).

Figure 4.

Histological scoring. (a) Residual material area in each group at 4, 8, and 20 weeks after surgery and (b) new bone formation area in each group at 4, 8, and 20 weeks after surgery. *P > 0.05 compared with the PDLLA/HA group; **P < 0.05 compared with the PDLLA/HA group. PLGA/HA, poly(d,l-lactide-co-glycolide)/hydroxyapatite; PDLLA/HA, poly(d,l-lactic acid)/hydroxyapatite.

Discussion

As a hot research topic in oral biomaterials, PLGA/HA and PDLLA/HA have received substantial attention from many scholars. However, only a few studies have compared the differences in their characterization and in vitro culture, and there is a lack of in vivo experiments of direct implantation.¹⁹ Both PLGA and PDLLA are common bone defect repair materials available in the market. Because they only have bone conductivity and lack the ability to induce bone formation, they are often used as carriers of various biological factors.²⁰ Several studies demonstrated that polyester/HA composites are superior to single ceramic or polymer materials in terms of mechanical properties and biocompatibility.^21,22 Moreover, HA neutralizes the acidic environment generated by the degradation of polymers and reduces their autocatalytic effect, avoiding the occurrence of aseptic inflammation.²⁰ In this experiment, PLGA/HA and PDLLA/HA bone repair scaffolds were prepared through 3D printing. Because of the differences in their characteristics, including composition, degradation rates, and biocompatibility, the effects of these materials on bone defects might also vary in vivo. This experiment demonstrated the performance of both scaffolds and provided a more suitable repair material for bone defect repair.

There have been similar studies on bone defect repair using bone scaffolds, which can provide us with a reference. Zhao et al. prepared PDLLA scaffolds using the salt immersion method and implanted them into radial defects in rabbits, and the results illustrated that new bone mainly formed at the edge of the defect at 4 weeks. At 8 weeks, new bone formation began to appear in the central area of the defect, but there was no significant explanation of the material. Until 24 weeks, the broken ends remained visible, and more cavities were observed at the defect center of the PDLLA stent. The authors suggested that this could be attributable to the rapid degradation of the material. Instead of the salt immersion method, the present study used 3D printing to prepare bone repair scaffolds with more uniform pores. At 4 weeks, new bone appeared in the central area in the PDLLA/HA and PLGA/HA groups. Concerning the degradation rate, the 3D-printed PLGA/HA scaffold in this study degraded more rapidly, and it had a higher defect healing rate than the PDLLA/HA group and traditional PDLLA scaffolds. The PLGA/HA scaffold exhibited obvious degradation behavior after 8 weeks, along with a disordered arrangement of new bone tissue, and the callus completely surrounded the wound. However, no obvious degradation behavior was observed in the PDLLA/HA scaffolds at 8 weeks. In addition, the new bone grew along the pores of the material, suggesting the degradation rate of the material was slower than the rate of bone regeneration. This resulted in stress shielding, which was harmful to the repair of bone defects.^23,24 After 20 weeks, the broken end of the PLGA/HA scaffold was no longer visible, whereas the cortical bone was completely connected, penetrating the bone marrow cavity. However, although the material of the PDLLA/HA scaffold lost its integrity at this time, the bone remodeling was not completed.

The residual material area and new bone formation area data illustrated that the repair of bone defects was significantly better in the PLGA/HA group than in the PDLLA/HA group. Existing studies indicated that the complete degradation time of PLGA (75:25) was 4 to 5 months, versus 12 to 16 months for PDLLA, consistent with our experimental results.^25,26

The underlying reasons for these results include both the different degradation rates and the higher hydrophilicity of PLGA. PLGA is a copolymer of poly(glycolic acid) (PGA) and PLA. Hydrophilicity and the ease of degradation increase as the proportion of PGA in the copolymer PLGA increases. This is attributable to the high polarity and good hydrophilicity of PGA.²⁷ PDLLA is a common form of PLA, and both polymers have one extra CH3 moiety in their structures than PGA. Thus, their polarity is reduced, and their hydrophilicity is lower than that of PGA and PLGA. Lee et al. inoculated chondrocytes on PLGA and PLA scaffolds and cultured them together for 3 weeks, finding that the levels of glycosaminoglycan and total collagen were significantly higher in the PLGA group than in the PLA group.²⁸ The researchers also revealed that the high hydrophilicity and rapid degradation rate of PLGA were attributable to its porous structure, which greatly enhanced the adhesion of chondrocytes. Because in vitro cell experiments cannot fully reflect the function of PLGA and PDLLA in vivo, we compared the two materials by implanting them into rabbit radii. Our study results were consistent with those of Lee et al.

In summary, PLGA/HA is more suitable for repairing bone defects than PDLLA/HA from the perspectives of new bone generation and material degradation. PDLLA/HA has an excessively slow degradation rate, which hinders the healing of bone defects to some extent. Although this hindrance might limit the application of PDLLA/HA in bone defect repair materials, it is still of great research value in fields in which mechanical strength needs to be maintained for a long time, including barrier membranes and fixation nails. Because of the lack of experiments on mechanical properties during material degradation in this study, certain limitations remain. In the future, further research will be conducted on the degradation and bone repair behavior of the two materials under in vivo stress.

Footnotes

Acknowledgements

The authors are grateful to the laboratory of the People’s Hospital of Inner Mongolia Autonomous Region for supporting this study.

Author contributions

S.L.: methodology, validation, formal analysis, and writing-original draft; X.L.: methodology, validation, and writing-original draft; J.S.: supervision and project administration; C.B. and B.F.: methodology and validation; P.Y. and W.Z.: data curation, methodology, and validation; J.Z. and J.L.: Micro-CT data collection and analysis; B.S.: supervision and project administration. All authors have read and agreed to the published version of the manuscript.

Data availability statement

The data are available from the corresponding author on reasonable request.

Declaration of conflicting interests

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Funding

This work was funded by the Science and Technology Program Foundation of Inner Mongolia Autonomous Region (2020GG0301), Inner Mongolia Medical University 2023 College Student Science and Technology Innovation Talent Cultivation Project(YCPY2023159), and Inner Mongolia Medical University Joint Project (YK2023LH075). The funders had no role in the study design, data collection, data analysis and interpretation, writing of the report, or the decision to submit the article for publication.

ORCID iD

Bo Shao

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