Effects of a novel biodegredable implant system on a rat tibia fracture model

Mehmet Yalçınozan; Mehmet Türker; Meriç Çırpar

doi:10.5152/j.aott.2020.18331

. 2020 Jul;54(4):453–460. doi: 10.5152/j.aott.2020.18331

Effects of a novel biodegredable implant system on a rat tibia fracture model

Mehmet Yalçınozan ¹, Mehmet Türker ^2,^✉, Meriç Çırpar ³

PMCID: PMC7444870 PMID: 32812878

Abstract

Objective

This study aimed to determine the effects of a novel biodegradable implant releasing platelet-derived growth factor (PDGF) at the fracture site on fracture healing in a rat tibia fracture model.

Methods

In this study, 35 male Sprague-Dawley rats weighing between 300 and 350 g were used. The rats were divided into four groups: Group A (control group without any treatment, n=10), Group B (spacer without PDGF Group, n=10), Group C (spacer with PDGF group, n=10), and Group D (healthy rat Group, n=5). Standardized fractures were created in the right tibias of rats, and then biodegradable implants made of poly-β-hydroxybutyrate-co-3-hydroxy valerate were implanted at the fracture sites in Groups B and C. In Group C, implants were loaded with 600 ng of PDGF. Animals were sacrificed 30 days after the operation, and fracture healing in each group was assessed radiologically based on the Goldberg score. Furthermore, the anteroposterior (AP) and mediolateral (ML) callus diameters were measured macroscopically, and fracture sites were mechanically tested.

Results

In the radiological assessment, Group C showed higher fracture healing rate than Groups A and B (p=0.001), whereas no significant difference was found between group C and Group D (p>0.05). In the macroscopic assessment, while Group C exhibited the thickest AP callus diameter (p=0.02), no significant differences in ML callus diameters existed among the groups (p>0.05). Mechanical testing revealed that Group C had higher torsional strength (p=0.001) and stiffness than Groups A and B (p=0.001) while there was no significant difference between Groups C and D (p>0.05).

Conclusion

Biodegradable implant releasing PDGF may have positive effects on fracture healing.

Keywords: Biodegradable, Continuous releasing systems, Implants, PDGF, Fracture healing

With the evolution of medical science, research on implant improvement has concentrated on biodegradable plastic materials (1, 2). The application of biopolymers has many advantages over most other materials, such as better biocompatibility and biodegradability and being environmentally friendly (3). Biodegradable polymers are being used in many areas of medicine, such as drug delivery systems, tissue engineering, and other applications (2).

Many conditions, such as trauma, tumor resection, deformity surgeries, infections, etc., may cause bone defects. Bone tissue has some regeneration and remodeling capacity provided by the natural healing process. However, the loss of bone tissue may exceed the repair capacity of the bone (4). As autologous bone grafting is still the gold standard procedure in such cases, the need for additional surgery and its possible complication risks are some of the limiting factors in its frequent use (5). Therefore, regenerative medicine using intracellular and extracellular signaling pathways has become important for overcoming these limitations (6). There are identified factors that have a role in fracture healing with their local or systemic effects. One of those factors is platelet-derived growth factor (PDGF), which has a powerful effect on mesenchymal cells (7–9). One of the known effects of PDGF on bone tissue is osteoblastic migration (10). In addition to the chemotactic feature, it is a mitogenic factor and has been shown to stimulate mesenchymal cells that initiate chondrogenesis and bone formation (11–13). The expression of PDGF at the fracture site also stimulates osteoprogenitor cell differentiation and collagen synthesis. In addition to these osteoblastic effects, PDGF also accelerates bone resorption by increasing the number of osteoclasts at the fracture site and provides remodeling (14, 15). PDGF is formed by two different polypeptide chains (A and B) and named after the combinations of these chains (PDGF-AA, PDGF-AB, PDGF-BB) (16). A multicenter study revealed that better success with comparable fusion rates, less pain and fewer complications can be achieved with a local PDGF-BB application after ankle joint fusions compared with autogenous bone grafting (17). The aim of our study was to research the effects of a localized PDGF release from poly-β-hydroxybutyrate-co-3-hydroxyvalerate (PHBV)-based implants at the fracture site. With the application of a successful growth factor-releasing biodegradable implant, the local application of a growth factor that has positive effects on fracture healing will noticeably improve treatment efficacy as healing occurs earlier.

Materials and Methods

Groups

Ethics approval for this study was obtained from the Experimental Animal Ethical Committee of Kırıkkale University School of Medicine. The study was performed on 35 rats, which were divided into 4 groups. A fracture model was performed on the right tibias of all rats. Group A (n=10) was the control group of this study, and these rats did not receive any treatment. Groups B (n=10) and C (n=10) were the treatment groups, and biodegradable spacers were implanted at the fracture sites in these animals. Group C implants were loaded with 600 ng PDGF-BB, enough for a 20 ng per day release for 30 days (9). Group D (n=5) included healthy rats.

Implant preparation

Polymer (PHBV) was mixed in a glass beaker with chloroform until the solution became a homogenous and viscous paste. This homogenous solution was injected into a 3 mm diameter cylindrical mold (Figure 1). After 12 hours of waiting time, the plastic was hardened enough to be cut clearly. Two parts of the prepared PHBV cylinder measuring 40 mm and 42 mm were cut and used in the study. The 40-mm part was divided into 20 empty 3×2 mm cylindrical implants. The 42-mm implant was solved with chloroform, and 12.5 μg of PDGF-BB was added to the solution. This solution (PHBV+PDGF) was injected into the mold. After the hardening of the plastic cylinder, it was divided into 21 PDGF-BB-loaded (600 ng each) 3×2 mm cylindrical implants (Figure 2).

Figure 1. a–c — Sections of the implant preparation process; a. The polymer pellets inside the glass mortar before preparation, b. polymer paste after processing with chloroform, and c. the injection mold system

Figure 2. a, b — Polymer spacer measurements before application; a. the caliber of the spacers and b. the height of the spacers

Surgical procedure

All surgeries were performed under general anesthesia in sterile operating room conditions after proper surgical preparations. A combination of ketamine (Ketalar^®, Pfizer, Turkey) 50 mg/kg and xylazine (Rompun^®, Bayer, Turkey) 10 mg/kg was injected from the left inguinal area intraperitoneally to provide general anesthesia. The right legs of all the rats were shaved and prepared with 7.5% povidone-iodine (Batticon^®, Adeka, Turkey) preoperatively to lessen the risk of infection. An anteromedially longitudinal incision of approximately 1 cm was made at the proximal cruris. The fracture model was produced at the proximal metaphysodiaphyseal region after the dissection of soft tissues (Figure 3a) with a 3.0 mm sterile drill at a speed of 120 rpm (18, 19) (Figure 3b). Incisions in Group A rats were closed after achieving the fracture model. Group B and C rat tibias were drilled, and implants were applied at the fracture sites (Figure 3. c, d). The incisions were sutured anatomically with 4/0 absorbable braided sutures and cleaned with a 7.5% povidone-iodine solution. The study ended with the sacrifice of all the rats on the 30^th day postoperatively. No complications were observed at the time of the study. Rats were sacrificed by carbon dioxide inhalation. Tibias were harvested subsequent to the radiologic evaluation, and during preparation they were kept in a 10% formaldehyde solution.

Figure 3. a–d — Intraoperative photos representing (a) the dissected medial tibial cortex, (b) the drilling of the tibia, (c) the implant placement, and (d) the tibial fracture model with an implant in place before skin closure

In this study, we examined the tibias radiologically, macroscopically, histologically, and mechanically.

Radiological evaluation

Group A (n=10), B (n=10), and C (n=10) rats were sacrificed on the 30^th postoperative day. All the relevant x-rays were taken immediately after surgery (day 0) and sacrifice (day 30), and their Goldberg scores were evaluated (20). This scoring system gives 0 points for nonunion, 1 point for the possibility of union, and 2 points for completely united fractures. Three blinded researchers (MY, MT, MC) evaluated all the radiological images independently according to the scoring system. When there was a disagreement about the scores, reevaluation was made until a consensus was reached to prevent bias.

Macroscopic evaluation

All the tibias were harvested after the radiological evaluation. The AP and ML diameters of callosities were determined and noted at their largest site with a digital caliper (PMS 150, Conrad Electronic Gmbh., Germany).

Mechanical testing

All the tibias were examined mechanically with special torsion testing software (Partner™, Instron Inc., USA) using a low capacity torsion testing system (Instron 55MT2, Instron Inc., USA). A sliding mold frame was prepared to center the bones in the mold. This system ensured centralization of the bone, eliminating the potential bending and shear forces during the test (Figure 4). The bones were molded into acrylic cement applied to the dynamic and static chins of the system and only the callus was left outside the cement to undergo the torsional forces at the fracture site (Figure 5). Torsional tests were performed at a constant speed of 2.5 deg/sec. The last highest value in the load-degree graph was recorded as the maximum torsional strength (N.m) (Figure 6). This value was then divided by the degree at which the fracture occurred to calculate the torsional stiffness value. These two values were used to compare the groups.

A sliding mold system that has been used to center the bones in the mold until satisfactory hardening of the cement is achieved

Tibias buried inside cement molds before mechanical testing

Sample torque/angle graph obtained from the mechanical testing software

Histological evaluation

Two random tibia samples of Groups A, B, and C that were eligible were evaluated histologically for fracture healing and callosities after mechanical tests. The bones were fixed in a 10% formaldehyde solution and stayed in 10% nitric acid/formalin solution for 24 hours for decalcification. They were cut into 1 mm thick sections and stained with hematoxylene/eosin. The evaluation was performed with a scoring system defined by Huo et al. (21) (Table 1).

Table 1.

Histological scoring system

Score	Histological findings
1	Fibrous tissue
2	Mostly fibrous, less cartilaginous tissue
3	Equal amount of fibrous and cartilaginous tissue
4	Cartilaginous tissue
5	Mostly cartilaginous tissue, less immature (woven) bone
6	Equal amount of cartilaginous tissue and immature bone
7	Mostly immature (woven) bone, less cartilaginous tissue
8	Immature (woven) bone
9	Mostly immature, less mature bone
10	Mature (lamellar) bone

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Statistical analysis

One-way analysis of variance (ANOVA) was performed followed by Tukey post hoc tests to identify differences between treatment groups for quantitative values, and a Chi-square test was performed for qualitative values. (Statistical Package for Social Sciences version 17.0, SPSS Inc.; Chicago, IL, USA) Significance was established at p<0.05.

Results

Radiological healing

X-rays taken on the 30th day postoperatively showed significantly better radiological healing in Group C than in the other treatment groups. (p=0.001). The difference in radiological healing between Group A and Group B was not significant (p=0.293) (Table 2 and Figure 7).

Table 2.

Goldberg scores on the 30^th postoperative day

Goldberg scores	Ave± SD	Min	Max
Group A	0.7±0.31	0	1
Group B	1.4±0.56	0	2
Group C	1.8±0.31	1	2
p=0.293^*		p=0.001^**

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Ave: average; SD: standard deviation; Min: minimum; Max: maximum

p value of the radiological comparison between Group A and Group B Goldberg scores. (Chi-squared test)

^**

p value of the radiological comparison between Group C and Group A Goldberg scores. p value of the radiological comparison between Group C and Group B Goldberg scores (Chi-squared test)

Figure 7. a–c — Sample radiographs of (a) the untreated group (Group A), (b) the treatment group with blank implants (Group B), and (c) the PDGF-releasing implant group (Group C)